RE: [Histonet] bone paraffin embedding
Nicole, You can very easily fix the bone in 10% NBF and then go into your decalcification process. Remember that fixation rate of bone is generally around 1mm per 24 hours (in all directions) and that it is good to have a minimum of 20:1 ratio of solutions to specimen size for each step. I would also recommend either 5% or 10% formic acid for decalcification. Can you tell us more specifics about the bone and what you wish to accomplish histologically? This information would be I might also suggest contacting Sarah Mack (copied to this message) from the University of Rochester. She is the new Hard Tissue Committee Chairperson for the National Society for Histotechnology and an expert in decalcified bone. I might also add that Sarah and Kim Simmons, NSH Education Development Manager, are working on educational opportunities and resource materials for those working with bone, biomaterials and medical device implants. If you are not a member of the NSH then you might want to consider becoming a member www.nsh.org/content/benefits-membership so that you can be kept up to date with what's coming soon from the Hard Tissue Committee. Best Regards, Jack Date: Mon, 3 Nov 2014 09:28:01 -0600 From: ncose...@siumed.edu To: histonet@lists.utsouthwestern.edu Subject: [Histonet] bone paraffin embedding Histonetters: Our lab needs to paraffin embed and cut bone. Is there a special process for fixation of bone, or can it be harvested and dropped right into NBF? -- Nicole Cosenza Research Technician Institute for Plastic Surgery SIU School of Medicine Springfield, Il 217.545.3862 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] bone paraffin embedding
Sorry Nicole I forgot to finish a thought before I hit send. What I was going to say was any additional information you could provide regarding the specimen and what you wanted to see at the microscope can sometime yield additional information regarding specific processing protocols, staining, tips and tricks, etc. Best, Jack From: ratliffj...@hotmail.com To: ncose...@siumed.edu; histonet@lists.utsouthwestern.edu CC: sarah_m...@urmc.rochester.edu; k...@nsh.org; ratliffj...@gmail.com Subject: RE: [Histonet] bone paraffin embedding Date: Mon, 3 Nov 2014 12:07:06 -0500 Nicole, You can very easily fix the bone in 10% NBF and then go into your decalcification process. Remember that fixation rate of bone is generally around 1mm per 24 hours (in all directions) and that it is good to have a minimum of 20:1 ratio of solutions to specimen size for each step. I would also recommend either 5% or 10% formic acid for decalcification. Can you tell us more specifics about the bone and what you wish to accomplish histologically? This information would be I might also suggest contacting Sarah Mack (copied to this message) from the University of Rochester. She is the new Hard Tissue Committee Chairperson for the National Society for Histotechnology and an expert in decalcified bone. I might also add that Sarah and Kim Simmons, NSH Education Development Manager, are working on educational opportunities and resource materials for those working with bone, biomaterials and medical device implants. If you are not a member of the NSH then you might want to consider becoming a member www.nsh.org/content/benefits-membership so that you can be kept up to date with what's coming soon from the Hard Tissue Committee. Best Regards, Jack Date: Mon, 3 Nov 2014 09:28:01 -0600 From: ncose...@siumed.edu To: histonet@lists.utsouthwestern.edu Subject: [Histonet] bone paraffin embedding Histonetters: Our lab needs to paraffin embed and cut bone. Is there a special process for fixation of bone, or can it be harvested and dropped right into NBF? -- Nicole Cosenza Research Technician Institute for Plastic Surgery SIU School of Medicine Springfield, Il 217.545.3862 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Protocol of MMA Plastic section TRAP staining
Hello Dorothy! I will forward you my protocol just as soon as I can get to my computer. On a side note, I no longer Chair the Hard Tissue Committee for the National Society for Histotechnology (NSH) as I have now been elected to the NSH Board of Directors as the Region III Director for the Southeast region. With that said, Sarah Mack is now the new Hard Tissue Committee Chairperson and Kim Simmons is the Education Development Manager for the NSH. I tell you this because Sarah is located in your part of the country and is currently the Histology Core Manager at University of Rochester Medical Center, Center for Musculosketeal Research. She works with bone and will be an good resource for you. I have copied her on this message. Kim Simmons will be helping to organize bone related educational materials and events for the NSH in the next year so she will be another good reference for you to follow. I have copied her to this message as well. I will still be a part of the Hard Tissue Committee in a supporting role. I just wanted you to know your additional options for information related to the histology of bone, biomaterials and medical device implants. Best Regards, Jack Ratliff On Oct 22, 2014, at 3:20 PM, Dorothy Hu abt...@gmail.com wrote: Dear Histoneters, Can someone email me your TRAP protocol for MMA section? I have one, but staining is not consistent. No matter I stain for how long, ~ several hours sometimes. There are always variations. I sincerely hope I can get different protocol form you and make change. I remember saw other TRAP protocol which is much longer than mine. The protocol I used was: 1. Dissolve 12mg Naphthol AS-MX phosphate (Sigma, N-5000) in 0.5ml N,N methylformamide (Sigma,D8654). 2. Mix above into 50 ml Sodium tartrate with 0.1M sodium acetate buffer at pH5.0. 3. Mix 30mg Fast Red Violet LB salt (Sigma, F3381) with above solution and filter. Stain slides @37oC for at least one hour. The solution can be use for one month when stored in 4oC. Continue to do counterstain and mount slides. Thank you. Dorothy Hu MGH Endocrine histocore Email: d...@partners.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Blades for cutting resin on a microtome
Veronique, May I ask what type of specimen is embedded into the JB-4 resin? Nevertheless, you should be able to cut these blocks using a tungsten-carbide knife. While there are a few vendors out there that sell these knives, in my laboratory I personally use knives re-sharpened by Delaware Diamond Knives (DDK). Please feel free to message me privately if you need further assistance as I have been working with resin embedded specimens for over 17 years. I will also encourage you to reach out to Sarah Mack as she is the new Hard Tissue Committee Chairperson for the National Society for Histotechnology. You can find her contact information and additional information about the committee by visiting www.nsh.org! Best Regards, Jack Ratliff On Sep 12, 2014, at 9:32 AM, Véronique Barrès veronique.bar...@gmail.com wrote: Happy Friday Histonetters! I am working on a histology platform in a research center and someone came to me last week and asked to cut blocs of resin (JB-4 resin) on the microtome. I never cut anything else than paraffin, so I was wondering if some of you had advices for me? They never did it neither and took their protocol in a paper where it was said that we should use disposable glass knife instead of standard metal blades. Are any of you ever used those knife? Where do you buy them? We have an old Leica RM2125. Thanks for your advices! Véronique ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Bone Histology Protocol
Trevor, May I ask first if there is a particular reason why you are wanting to decalcify? Have you ever considered resin/plastic embedding of non-decalcified bone? Also, what type/species of bone are you wanting to process? My name is Jack Ratliff and I Chair the Hard Tissue Committee for the National Society for Histotechnology and as a professional histology organization and committee focusing on bone, biomaterials and medical device implants, we offer educational solutions to help those like yourself that are in search of information. This is accomplished through the presentation of workshops at the state, regional and national levels or even by providing free reference materials like processing manuals, books for purchase at non-member or member discounts, and free access to the archives of The Journal of Histotechnology to society members. If any of this interests you please check out the NSH website (www.nsh.org) where you can navigate around to view all of what the society has to offer, become a member and even connect with the Hard Tissue Committee. One last thing I can tell you now is that there will be several bone workshops available at the NSH Symposium/Convention this August in Austin, TX. I will also be one of the speakers at this meeting giving a workshop on resin embedding techniques in support of bone, biomaterials and medical device implants. Best Regards, Jack On Feb 28, 2014, at 10:40 PM, Wait, Trevor Jordan wa...@livemail.uthscsa.edu wrote: Hello colleagues! I'm currently trying to construct a complete Bone Processing Protocol that includes fixation (10%NBF), decalcification (EDTA), Dehydration, Clearing of the decalcificant (Xylene), infiltration, and Embedding in Paraffin. I would like to look at some procedures just to get a good backbone of what a complete procedure is displayed like and I was hoping somebody could give me a website or a source where I could see some. I'm kind of new to bone histological processing so any procedures that are reliable will help! Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate Abilene Christian University Class of 2013 Graduate B.S. Biochemistry ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Where do you order MMA for undecalcified plastic sectioning mouse bone?
Dorothy, I personally order from Sigma and I have to tell you that in all my years of working with methyl methacrylate, they have been the most reliable source. I must admit that I did once use Fisher for a brief period of time due to pricing concerns from the purchasing department as there was something of an issue with the hazardous shipping from Sigma being almost as much as the MMA price, but not the case with Fisher. Honestly though I only used Fisher for about a year to a year and a half and switched back because of an issue with a particular lot that I encountered. During that particular issue my MMA blocks had polymerized with a sort of amber tint and with a pleasant orange fruity smell. It was alarming to me because I was completely used to having made crystal clear resin blocks and now I was starting to really love this orange smell during block preparation at the grinder that used to be a pungent MMA smell thats not really good for you under prolonged or excessive exposure. As time went by I then started to notice a change in polymerization rates and even sometimes an incomplete polymerization with a rubbery surface. While I did not recall any serious issue with staining, I did notice that cutting thin sections became a bit inconsistent and troublesome at times. It was then that I needed to discover if it was something that I was now doing differently than in the several years past of consistency or the chemicals that I was using. I ordered a new bottle off MMA from Sigma, changing only one variable, and ran a test. Turned out that I was back instantly to the clear blocks that I was accustomed to creating and that the issue had to be the Fisher bottle/lot of MMA. I want to point out that I am NOT saying that the Fisher MMA is an unreliable source of MMA. It was in my opinion clearly an issue of a bad lot/batch of product. However, given my OCD tendencies within the laboratory, I quickly reminded myself that if it's not broke, don't fix it! Basically, I shouldn't let pricing sacrifice the quality and consistency that I was accustomed to experiencing as I only switched suppliers due to a request for a cheaper alternative by the purchasing department. Oh and one more thing, I found out later that the orange smell was due to the presence of either solution or residue from the orange cleaning solution that the glass bottles are subjected to prior to filling with chemicals so like I said, could have been a one in a million experience but my mind told me I could not take any more chances! :) Best Regards, Jack From: dml...@gmail.com Date: Fri, 31 Jan 2014 12:56:55 -0500 To: abt...@gmail.com Subject: Re: [Histonet] Where do you order MMA for undecalcified plastic sectioning mouse bone? CC: histonet@lists.utsouthwestern.edu Hi Dorothy, I buy Methyl Methacrylate from Fisher Scientific. -Damien L. Sent from my iPhone On Jan 31, 2014, at 12:42 PM, Dorothy Hu abt...@gmail.com wrote: which company do you ordered MMA? Sigma has back order, so I try to find reliable resource here. Thanks, Dorothy abt...@gmail.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] zebrafish embryos histology
Patty, I have personally never performed histology on zebra fish embryo's, but if I was to test it out on my own I might try using the JB4 Plus GMA kit. It just seems to me that paraffin might cause too much shrinkage and maybe using an MMA protocol might be too harsh on the tissue. Besides, given the hydrophilic nature of GMA it just might be the best overall solution. Best, Jack From: pl...@uab.edu To: histonet@lists.utsouthwestern.edu Date: Wed, 22 Jan 2014 15:00:40 + Subject: [Histonet] zebrafish embryos histology Can anyone give me a method for zebra-fish embryo histology? The papers I've read show photos, but no description of histology in M M. I need to put several embryos in each block, and get the orientation correct, and put multiple sections on each slide, in hopes of getting one or two that are perfect. Any suggestions would be greatly appreciated. Thanks, Patty Lott UAB CMBD Core Lab 205-934-2007 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Do I have to destabilize MMA?
Dorothy, There are some that completely believe that it is necessary to destabilize MMA prior to use and they are not necessarily wrong as the protocol has worked for them without issue..so I assume. I personally have NEVER had to or tried this destabilization method, quite frankly because I have NEVER had a problem creating an acrylic resin embedded block, regardless of the tissue or size of the tissue, when simply combining monomer (MMA) with softener (DBP) and catalyst (Perkadox 16) in my almost 15 years of exclusively working with this resin for demonstrating bone, biomaterials and medical device implants. Again, I am not saying that the protocol given to you does not work, but rather it is my personal experience that this destabilizing method is an unnecessary step and a waste of time and expense. Hope this helps and please feel free to contact me if you have any additional questions or concerns. Best Regards, Jack Ratliff On Jan 15, 2014, at 10:02 PM, abt...@gmail.com wrote: I am new to MMA plastic bone technique. Some one gave me his protocol, in which has NaOH and d-water to wash MMA mixture before drying it in CaCl2. But others told me I don't need to do the destabilization step. Could any expert in this area to tell me if this step is necessary? And why have to do? Sent from my iPad ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Undecalcified sample in paraffin and plastic media
Rui, You will definitely want to consider using plastic media like methyl methacrylate (MMA). It will cause less shrinkage in the tissue during polymerization, you can still cut at a range of 4-12 microns using a rotary microtome and tungsten-carbide knife, any mineralization present in the tissue will infiltrate and polymerize well allowing for enhanced stabilization of tissue and section morphology throughout microtomy, and you can even deplastify the sections with certain MMA formulations to increase staining options. Please let me know if you do wish to continue with plastic media as I have helped many labs to get started with and/or to refine their current capabilities with MMA. Additionally, I would like to point out that I Chair the Hard Tissue Committee (HTC) for the National Society for Histotechnology (NSH). Membership with the NSH has several benefits that could also help you to move forward with your project at your own pace. For example, as a member you will have access to all archived publications of the Journal of Histotechnology (JOH). With this access to the JOH via Manny Publishing, the HTC has created a reference document that collates all relevant publications (1970's to present) that pertain to bone, biomaterials, medical device implants, resin histology, etc., so that one can easily locate and obtain publication information relevant to their niche specific needs. Rest assured that I will be happy to help you either way you choose to move forward. Best Regards, Jack On Sep 23, 2013, at 9:19 PM, Rui TAHARA ru...@hotmail.com wrote: I have undecalcified paraffin embed samples that were sectioned at 10 micron that I want to stain with Von kossa. Because samples are embryonic quail heads (ossification starts to happen) and still soft enough to section with standard rotary microtome with tungsten knife in paraffin. My intention is to 3D reconstruct anatomies based on histological sections. Because of this, I am wondering if I should actually use plastic media rather than paraffin to keep the section shape as consistent as possible. Does plastic embed material actually preserve the consistent shape among sections better than paraffin embed sample? No winkle etc..? Is there any other advantage that I actually should use the plastic media than paraffin for what I want to do? I know downside of plastic media is that in general plastic embedding process are lengthy and plastic embedding material are expensive than the paraffin ones, and are mainly use for bone to support the hard material for sectioning. When I sectioned some ossified samples, beak start to fall off from section and the section show the lines from the possibly scratched knife. Is this indication of paraffin media that does not provide enough strength for sectioning? I thought it may possibly the poor infiltration. In our lab nobody has processed the plastic embedding and sectioning (we have only standard microtome, no vaccum machine. Can I section plastic embed sample with the standard microtome at 10 micron?) so I would like to have any input before actually making a plastic embed sample. Any suggestions would be appreciated. Rui TAHARA Biology Department McGill University ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Microtome advise...
How about the new Sakura with the auto orientation feature! I saw it on display at the Missouri Society for Histotechnology meeting and it was quite impressive! Of course if you have the budget, there's alway the non-contact laser microtome from Rowiak! I have seen and used that unit to cut fresh soft tissues (brain) and resin embedded bone and biomaterial implants! Jack On Jul 18, 2013, at 7:57 AM, Tom McNemar tmcne...@lmhealth.org wrote: Hello all. I am preparing my budget and am seeking recommendations on automated microtomes. I am specifically interested in brands other than Leica (I already have two). I would appreciate any thoughts you may have. Thanks. Tom McNemar, HT(ASCP) Histology Co-ordinator Licking Memorial Health Systems (740) 348-4163 (740) 348-4166 tmcne...@lmhealth.orgmailto:tmcne...@lmhealth.org www.LMHealth.orgfile:///C:\Documents%20and%20Settings\TMCNEMAR\Application%20Data\Microsoft\Signatures\www.LMHealth.org This e-mail, including attachments, is intended for the sole use of the individual and/or entity to whom it is addressed, and contains information from Licking Memorial Health Systems which is confidential or privileged. If you are not the intended recipient, nor authorized to receive for the intended recipient, be aware that any disclosure, copying, distribution or use of the contents of this e-mail and attachments is prohibited. If you have received this in error, please advise the sender by reply e-mail and delete the message immediately. You may also contact the LMH Process Improvement Center at 740-348-4641. E-mail transmissions cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Microtome advise...
BTW. I'm certain that the Sakura unit will be on display at the NSH S/C meeting in Providence, RI (20-25 Sept 2013) and I know for a fact that the laser microtome will be on display there as well because it will be demonstrated and used, along with a Leica rotary microtome for part of a workshop I am co-presenting with Bob Skinner (WS # 61 - A Detailed Examination of Working With Decalcified and Undecalcified Bone In Support of Preclinical and Clinical Research). Jack On Jul 18, 2013, at 8:55 AM, Jack Ratliff ratliffj...@hotmail.com wrote: How about the new Sakura with the auto orientation feature! I saw it on display at the Missouri Society for Histotechnology meeting and it was quite impressive! Of course if you have the budget, there's alway the non-contact laser microtome from Rowiak! I have seen and used that unit to cut fresh soft tissues (brain) and resin embedded bone and biomaterial implants! Jack On Jul 18, 2013, at 7:57 AM, Tom McNemar tmcne...@lmhealth.org wrote: Hello all. I am preparing my budget and am seeking recommendations on automated microtomes. I am specifically interested in brands other than Leica (I already have two). I would appreciate any thoughts you may have. Thanks. Tom McNemar, HT(ASCP) Histology Co-ordinator Licking Memorial Health Systems (740) 348-4163 (740) 348-4166 tmcne...@lmhealth.orgmailto:tmcne...@lmhealth.org www.LMHealth.orgfile:///C:\Documents%20and%20Settings\TMCNEMAR\Application%20Data\Microsoft\Signatures\www.LMHealth.org This e-mail, including attachments, is intended for the sole use of the individual and/or entity to whom it is addressed, and contains information from Licking Memorial Health Systems which is confidential or privileged. If you are not the intended recipient, nor authorized to receive for the intended recipient, be aware that any disclosure, copying, distribution or use of the contents of this e-mail and attachments is prohibited. If you have received this in error, please advise the sender by reply e-mail and delete the message immediately. You may also contact the LMH Process Improvement Center at 740-348-4641. E-mail transmissions cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Mixing Paraffin Brands
Pedro, I have been using a superb method from Bob Skinner for all my decalcified bone work. Basically, I infiltrate with 50% TissuePrep from Fisher Scientific and 50% EM400 from Leica. I then embed in straight EM400. I personally feel that I get good infiltration and I never have had a problem with my bone cutting. (knocking twice on woodLOL) I might also mention that I decalcify with 5% formic acid and clear after dehydration using methyl salicylate instead of xylenes. Jack Jack L RatliffOwner/Histologist, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology From: jgarfi...@lifecell.com To: lou...@princeton.huntingdon.com; rjbu...@yahoo.com; lguern...@ucsd.edu; histonet@lists.utsouthwestern.edu Date: Wed, 19 Jun 2013 14:17:22 -0500 Subject: RE: [Histonet] Mixing Paraffin Brands CC: Pedro, I recommend Paraplast-Xtra. Regards, Jackie Jacqueline D. Garfield | Manager, Histology Main908.947.1100 Fax 908.947.1085 Direct 908.947.1182 Emailjgarfield@ lifecell.com www.lifcell.com LifeCell Corporation | One Millennium Way | Branchburg, NJ | 08876 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Louro, Pedro Sent: Wednesday, June 19, 2013 2:33 PM To: Rene J Buesa; Lucie Guernsey; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Mixing Paraffin Brands Just out of curiosity, what paraffin would people out there recommend using for animal bone joints and turbinates? We are currently using parapalst plus and was thinking of changing to a harder paraffin Any thoughts??? Pedro L. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa Sent: Wednesday, June 19, 2013 2:24 PM To: Lucie Guernsey; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Mixing Paraffin Brands Each paraffin has some additives to improve either its penetration rate or density to section. Mixing different melting points (MP) paraffin will result in another paraffin with an intermediate MP and the sectioning will be different. Preparing the block with the mixture will probably cause so troubles while sectioning. Why don't you just use them separate? There is no good reason to mix them and the two paraffins you mention are of good quality. René J. From: Lucie Guernsey lguern...@ucsd.edu To: histonet@lists.utsouthwestern.edu Sent: Wednesday, June 19, 2013 2:01 PM Subject: [Histonet] Mixing Paraffin Brands Hi, Does anyone know if there's a reason why one shouldn't mix different brands of paraffin? We normally use Fisherbrand's Paraplast Plus (melting point 56 C). We inherited about 8 bags of McCormick Paraplast X-tra (melting point 52 C). Will the different melting points be a problem? If we were to use the McCormick paraffin, the only place it may mix with the Fisherbrand paraffin is in the blocks themselves (as we refill the embedder). But I don't want to compromise the quality of our blocks just to not waste the free paraffin. Or, another option could be that we use the McCormick in the processor and the Fisherbrand in the embedder. Could that cause issues in the blocks as the tissue would be infiltrated with one brand and embedded in another? Maybe I'm over-thinking this.. Many thanks! Lucie Lucie Guernsey UCSD Dept. of Pathology lguern...@ucsd.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Our Values: Excellence, Drive, Ownership, Challenge, Teamwork, Respect LEGAL NOTICE This message is confidential and contains information which may be legally privileged. It is intended for the stated addressee(s) only. Access to this email by anyone else is unauthorised. If you are not the intended addressee, any disclosure or copying of the contents of this email or any action taken (or not taken) in reliance on it is unauthorised and is unlawful. If you are not the addressee, please inform the sender immediately and destroy the original message. The data in this email will have been screened for the presence of computer viruses known by the Company at the time the email was produced. The Company cannot guarantee, however, that the email or any attachments are virus free. Delivery of the email
RE: [Histonet] Paraffin processing native sheep ACL
You might also consider using methyl salicylate instead of xylenes. Thanks to the help of Bob Skinner, I have achieved very nice results with native tendon. Generally speaking these MS steps will take a little longer, but you can monitor the progress very easily by watching for complete transparency of the tendon. You can then even develop a somewhat standardized protocol if you plan to process this type of tissue in the future. You even have a lot more flexibility with MS than xylenes as prolonged use in xylenes can make the tissue more hardened and brittle. Lastly, it is not generally recommended to put MS on the tissue processor, so I process to the final 100% EtOH, perform the MS exchanges by hand, transfer the tissues into a manual wax step to get rid of as much MS as possible and then finish with three (3) automated wax steps on the tissue processor. For my wax infiltration I use a 50:50 blend of TissuePrep from Fisher Scientific and EM400 from Leica. I then embed in 100% EM400. Best Regards, Jack Jack L RatliffOwner/Histologist, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology From: a.pr...@tissueregenix.com To: histonet@lists.utsouthwestern.edu Date: Fri, 14 Jun 2013 07:45:47 + Subject: Re: [Histonet] Paraffin processing native sheep ACL CC: Hi Liz, I inherited the following protocol for ACL samples. It works quite well, but times probably could be reduced - the optimization is on my to-do list. 70% Alcohol - 1 hour 90% alcohol - 1 hour 100% Alcohol -2 hours 100% alcohol - 3 hours 100% alcohol - 4 hours Xylene - 1.5 hours Xylene - 1.5 hours Xylene - 3 hours Wax - 3 hours Wax - 3 hours Wax - 4 hours I cut the sections at 8um so they hold together better. Takes a while for all the wrinkles to disappear when floating out on water-bath so be patient Hope this helps. Andrew Andrew Prior Histologist Tissue Regenix Group E-mail: a.pr...@tissueregenix.commailto:a.pr...@tissueregenix.com Website: www.tissueregenix.comhttp://www.tissueregenix.com/ -- Message: 3 Date: Wed, 12 Jun 2013 16:59:52 -0400 From: Elizabeth Ronan lizro...@umich.edumailto:lizro...@umich.edu Subject: [Histonet] Paraffin processing native sheep ACL To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: 51b8e148.3020...@umich.edumailto:51b8e148.3020...@umich.edu Content-Type: text/plain; charset=ISO-8859-1; format=flowed Hello, I need to paraffin process native sheep anterior cruciate ligament (ACL) that has been fixed in 10% neutral buffered formalin for 7 days. I was wondering if anyone with more expertise on this subject could guide me with the best lengths of times in the alcohols, xylenes, and paraffin to fix ACL. I tried a 12 hour program but the sections crumbled in the middle and it appeared that the paraffin had not fully perfused the ligament. I have access to the following program, and can alter the lengths of the steps for as long as desired: Program: 70% 80% 95% 95% 100% 100% Xylene Xylene Paraffin Paraffin Paraffin Any advice is much appreciated. Thanks for your time, Liz The information transmitted is intended only for the person or entity to which it is addressed and may contain confidential and/or privileged material. Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by persons or entities other than the intended recipient is prohibited. If you received this in error, please contact the sender and delete the material from any computer. Although we routinely screen for viruses, addressees should check this e-mail and any attachment for viruses. We make no warranty as to absence of viruses in this e-mail or any attachments. Registered Office: The Biocentre, Innovation Way, Heslington, York, YO10 5NY Registered No. 05969271 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Bone, Biomaterials Medical Device Implants Histology Event In Cambridge, MA - May 4th, 2013
If you are currently working with bone, biomaterials or medical device implants, you won't want to miss this specialty histology event http://www.polysciences.com/Interactive-Histology-Forum-About/185/ sponsored by Polysciences Inc. and supported with six (6) continuing education units (CEU's) by the National Society for Histotechnology! There is still time to pre-register online http://events.r20.constantcontact.com/register/event?llr=gqebcrbaboeidk=a07e6rs5o33629b055e or onsite May 4th at the Hyatt Regency Cambridge http://www.polysciences.com/Interactive-Histology-Forum-Hotel-Travel/189/. Topics of discussion will include: A Closer Look at Fixation, Decalcification and Processing Techniques for Paraffin Embedded SpecimensPresented by Robert A. Skinner, HTL (ASCP), Director of Laboratory Resources, Center for Orthopaedic Research, University of Arkansas for Medical Sciences Histology Technique Selection and Challenges for Biomaterial and Medical Device Implanted TissuesPresented by Philip Seifert, MS, HTL (ASCP), Principal Scientist, CBSET, Inc. Acrylic Resins: A Practical Approach for Demonstrating Undermineralized Bone, Biomaterials and Medical Device ImplantsPresented by Jack L. Ratliff, BA, Owner, Ratliff Histology Consultants Chairman, Hard Tissue Committee National Society for Histotechnology The Use Of A Non-Contact Laser To Collect Thin Sections From A Variety of Soft And Hard Histological Tissues, Synthetic Bio-Resorbable Materials, and Tissues Containing Non-Resorbable Medical Device ImplantsPresented by Heiko Richter, Ph.D., Sales Product Management, Rowiak GmbH Introduction to Routine Staining and Special Staining with Plastic Resin TechniquesPresented by Valantou Grover, HT (ASCP) HTL, PA, Product Line Manager, Business Manager, Histologist at Polysciences, Inc. If you have any questions, please feel free to contact myself or Ashley Gidzinski (ashley.gidzin...@polysciences.com). We look forward to seeing you in Cambridge, MA and May the 4th be with you! Best Regards, Jack Owner/Histologist, Ratliff Histology Consultants, LLC Chairman, Hard Tissue Committee - National Society for Histotechnology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] GSH update
If you're a local Histotech or Pathologist within a 2 1/2 hour drive of Jekyll Island, you still have time to catch the second workshop of the day (1:30pm EST)Laser Microtomy: The Future of Soft Hard Tissue Histology! You won't want to miss this introduction to the use of femtosecond lasers to section fresh and resin embedded tissues! Jack On Apr 12, 2013, at 10:39 AM, Zimmerman, Billie bzimm...@gru.edu wrote: HT review is in session now. The students are very fortunate to have Robert Lott as an instructor. I have to confess I had about a dozen raw oysters last night at SeaJays but did cold coke chasers to kill the germs. We sat outside on the veranda and the weather was perfect. Vendors are setting up and we are looking forward to the vendor reception tonight. Sent from my iPhone Augusta State University and Georgia Health Sciences University have consolidated to become Georgia Regents University. Effective January 9, 2013, my email address has changed to bzimm...@gru.edu. Please update your address book to reflect this change. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Updates on Jekyll Island...Procrastinators please read this ASAP
PLEASE! PLEASE! PLEASE! I want that room! Oh and since you mentioned speedos and crochet bikinis, you want me to bring my white mankini with the British flag on the front? Just kidding, I think I had better leave that at home! Jack Jack L. RatliffOwner/Histologist, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology 389 Nichol Mill LaneFranklin, TN 37067(317) 281-1975 (c)(615) 236-4901 (o)(615) 236-4962 (f)jratl...@ratliffhistology.com From: bzimm...@gru.edu To: histonet@lists.utsouthwestern.edu Date: Thu, 28 Mar 2013 18:57:46 + Subject: [Histonet] Updates on Jekyll Island...Procrastinators please read this ASAP If you haven't reserved your room for the upcoming GSH meeting next month, please note that all island and standard Oceanside rooms have been reserved. But, just because you snoozed, were waiting on money for your institution, or you're just a classic procrastinator, there are still options. There are upgraded rooms such as the lanai. This room has a Jacuzzi, microwave, King size bed, small refrigerator, and a private balcony or patio. There's also the efficiency which has the kitchenette with a stove top, refrigerator, two double beds, sitting area, and private balcony or patio. The symposium rate still applies and if you have issues or concerns, contact Linda Schepps at Oceanside. She can't cure procrastination but she can help secure a room for you. While you're strolling around the historic area of the island in your speedo or crochet bikini, check out Becky's famous chicken salad. It's a little place with outside tables. Just walk up to the window and place your order. I attempted to have a chicken salad sandwich there but they were all sold out!! The lady managed to let me sample a teaspoon of it. It was delicious and didn't taste like that stuff in the grocery store. I plan to attempt another order when I return next month!! Augusta State University and Georgia Health Sciences University have consolidated to become Georgia Regents University. Effective January 9, 2013, my email address has changed to bzimm...@gru.edu. Please update your address book to reflect this change. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Updates on Jekyll Island
Im not that crazy Shirley, but I have worn a speedo on the beach within the last 8 months just for fun! LOL Yes, I have my reservation and I believe it is ocean/beach side, but that jacuzzi is a tempting thought to change! My Dad always says that only a fool wouldn't change their mind! LOL One more thing, if the weather is nice during those days, I'm seriously thinking of riding my Harley out there! See you all very soon! Jack From: powell...@mercer.edu To: ratliffj...@hotmail.com; bzimm...@gru.edu; histonet@lists.utsouthwestern.edu Date: Thu, 28 Mar 2013 16:15:51 -0400 Subject: Updates on Jekyll Island Waaay too much information Jack, just keep it covered. You can bring all those wonderful photos of your European trip with your Dad. I do hope you have made your reservations at Jekyll. Sounds like the rooms are going fast. See you at the beach. Oh and Jack, they will arrest you if you show too much. This is Georgia you know. Sp -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jack Ratliff Sent: Thursday, March 28, 2013 3:17 PM To: Zimmerman, Billie; Histonet Subject: RE: [Histonet] Updates on Jekyll Island...Procrastinators please read this ASAP PLEASE! PLEASE! PLEASE! I want that room! Oh and since you mentioned speedos and crochet bikinis, you want me to bring my white mankini with the British flag on the front? Just kidding, I think I had better leave that at home! Jack Jack L. RatliffOwner/Histologist, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology 389 Nichol Mill LaneFranklin, TN 37067(317) 281-1975 (c)(615) 236-4901 (o)(615) 236-4962 (f)jratl...@ratliffhistology.com From: bzimm...@gru.edu To: histonet@lists.utsouthwestern.edu Date: Thu, 28 Mar 2013 18:57:46 + Subject: [Histonet] Updates on Jekyll Island...Procrastinators please read this ASAP If you haven't reserved your room for the upcoming GSH meeting next month, please note that all island and standard Oceanside rooms have been reserved. But, just because you snoozed, were waiting on money for your institution, or you're just a classic procrastinator, there are still options. There are upgraded rooms such as the lanai. This room has a Jacuzzi, microwave, King size bed, small refrigerator, and a private balcony or patio. There's also the efficiency which has the kitchenette with a stove top, refrigerator, two double beds, sitting area, and private balcony or patio. The symposium rate still applies and if you have issues or concerns, contact Linda Schepps at Oceanside. She can't cure procrastination but she can help secure a room for you. While you're strolling around the historic area of the island in your speedo or crochet bikini, check out Becky's famous chicken salad. It's a little place with outside tables. Just walk up to the window and place your order. I attempted to have a chicken salad sandwich there but they were all sold out!! The lady managed to let me sample a teaspoon of it. It was delicious and didn't taste like that stuff in the grocery store. I plan to attempt another order when I return next month!! Augusta State University and Georgia Health Sciences University have consolidated to become Georgia Regents University. Effective January 9, 2013, my email address has changed to bzimm...@gru.edu. Please update your address book to reflect this change. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Bone processing help
Dr. De la Vega, My name is Jack Ratliff and I Chair the Hard Tissue Committee for the National Society for Histotechnology. I am not sure if anyone has provided you with a response to your message, but maybe I can be of assistance. Just so you know, the NSH prides itself on being an educational resource for those working in the field of Histology. The Hard Tissue Committee represents the educational arm of the society for those working with hard tissues like bone, biomaterials and medical device implants and especially those type of histological specimens embedded into resins or plastics! I would like to first direct your attention to the NSH website at www.nsh.org. If you go to the following link: http://www.nsh.org/content/benefits-membership, you will find information concerning membership benefits and how to join so that you can take advantage of what the society can do for you and your laboratory! Please visit this site first and then feel free to get back to me with any questions. As for your current issues, I can definitely help you with all that you are experiencing. At the moment I am currently away from the US and in Germany until the 26th of March working with a group (Rowiak GmbH - www.rowiak.de) that has developed a non-contact laser microtome (Tissue Surgeonhttp://www.rowiak.de/index.php?id=19) that can do what you are doing now and produce stained slides @ 10-15 microns in 30 minutes from a resin/plastic embedded block! If you can give me a few more hours, I will personally respond to your message privately. In the meantime, please visit the NSH website (www.nsh.org), consider its membership and also look to join the Hard Tissue Committeehttp://www.nsh.org/committee-detail/1044 so that I can provide you with meeting updates. With that said and seeing that you are in Massachusetts, on May the 4th, 2013, Polysciences, Inc. is hosting a full day educational event focusing on the *Histological Applications Techniques for Bone, Biomaterials and Medical Device Implants*. I will also be a speaker at this event. Those that attend can expect to further their knowledge, understanding and training of specialized histology techniques associated with bone, biomaterials and medical device implant specimen types and attendees will learn of the applicational relevance of the techniques used in the evaluation safety and efficacy of therapeutic treatments. Registration is currently open with an *Early Bird registration set to end this Friday, March 22nd*. The National Society for Histotechnology (NSH) will be providing 6 CEUs and Polysciences, Inc. will be providing a discount to any current NSH member as outlined below: *WORKSHOP FEES* * * *Before March 22, 2013: $149.00 for NSH Members / $199.00 for Non-Members* * * *After March 22, 2013: $179.00 for NSH Members / $229.00 for Non-Members* For the complete details of this full day *Histological Applications Techniques for Bone, Biomaterials and Medical Device Implants *event and to register online, please visit the following link: * http://www.polysciences.com/Interactive-Histology-Forum-About/185/* and sign up today to participate in the discussion of these specialized histology specimen applications and techniques that are rarely shared or even discussed on Histonet! You will not want to miss out on the information presented by 4 expert speakers in the field, all course materials, meals and a complete program book also containing technique specific protocols that you can repeat back in your lab! Best Regards, Jack Jack L. Ratliff Owner/Histologist, Ratliff Histology Consultants, LLC Chairman, Hard Tissue Committee - National Society for Histotechnology 389 Nichol Mill Lane Franklin, TN 37067 (317) 281-1975 (c) (615) 236-4901 (o) (615) 236-4962 (f) jratl...@ratliffhistology.com On Sun, Mar 17, 2013 at 8:09 PM, Jack Ratliff ratliffj...@hotmail.comwrote: Begin forwarded message: *From:* De La Vega Amador, Rodolfo Enrique rdelavegaama...@partners.org *Date:* March 15, 2013, 9:41:23 PM GMT+01:00 *To:* histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu *Subject:* *[Histonet] Bone processing help* Hi everybody, I am fairly new to all Histotechnology processes. We mainly work in our lab with mineralized bone with implants, so we fix them, dehydrate them, embed them in MMA, cut them, glued them to slides, ground them manually and then stain them. I need help in some parts of the process that need improvement: 1. We use Loctite 4471 on the samples, put them under vacuum, apply them to the slide and more vacuum. There's good results but there are some bubbles that still appear. Suggestions? 2. I can't seem to get the yellow/orange on bone with the Van Gieson stain. I've been doinga preheat at 55 °C, etching, rinse in DI water, Sanderson´s Rapid Bone Stain, running tap water, Van Gieson (commercial from DHM) for 30 seconds to 5 minutes, paper dry and quickly dehydrate with 100% EtOH. What
[Histonet] All Members Working with Bone, Biomaterials and Medical Device Implants
On May the 4th, 2013, Polysciences, Inc. is hosting a full day educational event focusing on the Histological Applications Techniques for Bone, Biomaterials and Medical Device Implants. On behalf of the speakers (Bob Skinner, Philip Seifert, Valantou Grover and Jack Ratliff) contributing to the educational content for this event, we would like for you to join us in Cambridge, MA at the Hyatt Regency Cambridge (overlooking Boston). With our combined histology experience of 90+ years in working with clinical and preclinical specimens pertaining to bone, biomaterials and medical device implants, you can expect to further your knowledge, understanding and training of specialized histology techniques associated with these specimen types and the applicational relevance of these techniques used in the evaluation safety and efficacy of therapeutic treatments. Registration for this event is now open with Early Bird registration set to end one week from Friday on March 22nd. The National Society for Histotechnology (NSH) will be providing 6 CEUs and Polysciences, Inc. will be providing a discount to any current NSH member as outlined below: WORKSHOP FEES Before March 22, 2013: $149.00 for NSH Members / $199.00 for Non-Members After March 22, 2013: $179.00 for NSH Members / $229.00 for Non-Members For the complete details of this full day Histological Applications Techniques for Bone, Biomaterials and Medical Device Implants event and to register online, please visit the following link: http://www.polysciences.com/Interactive-Histology-Forum-About/185/ and sign up today to participate in the discussion of these specialized histology specimen applications and techniques that are rarely shared or even discussed on Histonet! You will not want to miss out on the information presented by 4 expert speakers in the field, all course materials, meals and a complete program book also containing technique specific protocols that you can repeat back in your lab! We hope to see you in Cambridge (Boston) and May the 4th be with you! Best Regards,Jack Ratliff ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] teeth sectioning
Thanks for your message Wayne. I will definitely follow up with you upon my return! Please let me know if there is anything else that interest you with regards to Hard Tissue specimen types. I specifically work with the histology related to bone, biomaterials and medical device implants. In fact, I will be presenting on these topics at several histology meetings here in the U.S. throughout the year: Indiana Society for Histotechnology - Indianapolis, IN (March 8-9) - Technological Advancements in Microtomy: A Non-Contact Alternative to Conventional Histology Equipment Techniques Georgia Society for Histotechnology - Jekyll Island, GA (April 12-13) - Laser Microtomy: The Future of Soft and Hard Tissue Histology LINK: http://www.histosearch.com/gsh/symposium.html Polysciences, Inc. Histological Applications and Techniques for Bone, Biomaterials and Medical Device Implants - Cambridge, MA (May 4) - Acrylic Resins: A Practical Approach for Demonstrating Bone, Biomaterials and Medical Device Implants LINK: http://www.polysciences.com/Interactive-Histology-Forum-Agenda/187/ Missouri Society for Histotechnology - Columbia, MO (May 30 - June 1) - Technological Advancements in Microtomy: A Non-Contact Alternative to Conventional Histology Equipment Techniques LINK: http://www.nsh.org/content/missouri-society-histotechnology-msh National Society for Histotechnology Symposium/Convention - Providence, RI (Sept 20-25) - A Detailed Examination of Working with Decalcified and Undecalcified Bone in Support of Preclinical and Clinical Research (co-presenter w/ Robert Skinner) LINK: http://www.histoconvention.org Hopefully I will get to meet you at one of these upcoming meetings! Best Regards, Jack Date: Mon, 4 Mar 2013 19:47:35 +0800 From: e...@pigsqq.org To: ratliffj...@hotmail.com CC: turke...@gmail.com; histonet@lists.utsouthwestern.edu; jratl...@ratliffhistology.com Subject: Re: Re: [Histonet] teeth sectioning Jack, That sounds really awesome. I did some work with the teeth of sows (female pigs) from specimens collected at slaughter. Those are very difficult to decalcify, and when finished, are likely to have no nuclear detail remaining. Interested to hear what you learn Wayne Johnson Beijing Enable Ag Consulting Yuanmingyuan West Road Meiyuan Com, On 3:59, Jack Ratliff wrote: Mes, This is a very good question and I look forward to answers from individuals that have accomplished this with PMMA and a rotary microtome with tungsten-carbide knives. If you are talking about an undecalcified specimen embedded in PMMA, then I would imagine that the age of the rat could affect the ability to achieve an adequate infiltration of the resin. Again, I look forward to what others have to say about their success by the method you have outlined. On the other hand, I know you can achieve the micron thickness you desire if you were to use a non-contact femtosecond laser! The machine I am talking about is basically a laser microtome manufactured by Rowiak in Germany and it is officially called the TissueSurgeon. In fact, Dr. Heiko Richter from Germany has accomplished what you ask with human teeth, revealing the full anatomy of the tooth and even with ameloblasts on the enamel surface! I would be interested to hear more about your project. I will be traveling to Germany one week from today to work with this laser microtome until the end of the month so I could arrange to have laser cut sections made for you if you are interested and unable to make your cuts using PMMA and a rotary microtome. If you would like more information, please feel free to contact me by email reply. Best Regards, Jack Jack L Ratliff Owner/Histologist, Ratliff Histology Consultants, LLC Chairman, Hard Tissue Committee - National Society for Histotechnology On Mar 1, 2013, at 7:03 PM, mesruh turkekulturke...@gmail.com wrote: Dear Histonetters, I have one more question. Is it possible to obtain 5-10um thick sections of PMMA embedded teeth using regular Leica paraffin microtome (RM2265) equipped with disposable tungsten carbide blade? Thanks, Mes On Fri, Mar 1, 2013 at 7:40 PM, mesruh turkekulturke...@gmail.com wrote: Dear Histonetters, I am studying bone and teeth growth in rat maxilla. I will inject calcein green and would like to fix, embed and sections the rat maxilla. Any suggestions for the best method to fix, embed and section the samples for fluorescnet microscopy? Thank you very much! Mes HTL (ASCP) Memorial Sloan-Kettering Cancer Center On Fri, Mar 1, 2013 at 1:01 PM, histonet-requ...@lists.utsouthwestern.edu wrote: Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman
Re: [Histonet] teeth sectioning
Mes, This is a very good question and I look forward to answers from individuals that have accomplished this with PMMA and a rotary microtome with tungsten-carbide knives. If you are talking about an undecalcified specimen embedded in PMMA, then I would imagine that the age of the rat could affect the ability to achieve an adequate infiltration of the resin. Again, I look forward to what others have to say about their success by the method you have outlined. On the other hand, I know you can achieve the micron thickness you desire if you were to use a non-contact femtosecond laser! The machine I am talking about is basically a laser microtome manufactured by Rowiak in Germany and it is officially called the TissueSurgeon. In fact, Dr. Heiko Richter from Germany has accomplished what you ask with human teeth, revealing the full anatomy of the tooth and even with ameloblasts on the enamel surface! I would be interested to hear more about your project. I will be traveling to Germany one week from today to work with this laser microtome until the end of the month so I could arrange to have laser cut sections made for you if you are interested and unable to make your cuts using PMMA and a rotary microtome. If you would like more information, please feel free to contact me by email reply. Best Regards, Jack Jack L Ratliff Owner/Histologist, Ratliff Histology Consultants, LLC Chairman, Hard Tissue Committee - National Society for Histotechnology On Mar 1, 2013, at 7:03 PM, mesruh turkekul turke...@gmail.com wrote: Dear Histonetters, I have one more question. Is it possible to obtain 5-10um thick sections of PMMA embedded teeth using regular Leica paraffin microtome (RM2265) equipped with disposable tungsten carbide blade? Thanks, Mes On Fri, Mar 1, 2013 at 7:40 PM, mesruh turkekul turke...@gmail.com wrote: Dear Histonetters, I am studying bone and teeth growth in rat maxilla. I will inject calcein green and would like to fix, embed and sections the rat maxilla. Any suggestions for the best method to fix, embed and section the samples for fluorescnet microscopy? Thank you very much! Mes HTL (ASCP) Memorial Sloan-Kettering Cancer Center On Fri, Mar 1, 2013 at 1:01 PM, histonet-requ...@lists.utsouthwestern.edu wrote: Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman/listinfo/histonet or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... Today's Topics: 1. FNA Clia Guidelines (PicheGrocki, Jessica) 2. RE: FNA Clia Guidelines (Horn, Hazel V) 3. GSH Symposium April 12-14 (Zimmerman, Billie) 4. FSH abstracts deadline (Jerry Santiago, MSEd, HTL (ASCP) QIHC) 5. (no subject) (Vikrant Piprode) 6. The GSH 4oth Anniversary Meeting (David Kemler) 7. QIHC (Renee H. Workman) -- Message: 1 Date: Thu, 28 Feb 2013 20:26:30 + From: PicheGrocki, Jessica jpiche-gro...@wtbyhosp.org Subject: [Histonet] FNA Clia Guidelines To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 631955447a364b45b9458d2905635110655d2...@win08-mbx-01.wtbyhosp.org Content-Type: text/plain; charset=us-ascii Hi All, Quick questionwhat are the Clia requirements for Fine needle aspirate procedures? Is it considered high complexity testing? And who prepares the slides when the needle is handed off? Thank you, Jessica Piche, HT(ASCP) CONFIDENTIALITY NOTICE: This email and any attachments contain confidential information that is legally privileged. This information is intended only for the use of the individual or entity named above. The authorized recipient of this information is prohibited from disclosing this information to any other party unless required to do so by law or regulation. If you are not the intended recipient, you are hereby notified that any disclosure, copying, distribution or action taken in reliance on the contents of these documents is strictly prohibited. If you have received this information in error, please notify the sender immediately and delete these documents. Copyright (c) Waterbury Hospital -- Message: 2 Date: Thu, 28 Feb 2013 15:05:35 -0600 From: Horn, Hazel V hor...@archildrens.org Subject: [Histonet] RE: FNA Clia Guidelines To: 'PicheGrocki, Jessica' jpiche-gro...@wtbyhosp.org, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID:
Re: [Histonet] SRBS/van Gieson
What is it that you specifically want to accomplish that you can't do with a VonKossa/MacNeals Tetrachrome or Goldner's Trichrome Stain? Jack On Jan 18, 2013, at 1:51 PM, Herrick, James L. (Jim) herrick.ja...@mayo.edu wrote: Happy Friday Everybody, We are trying to develop a protocol for an SRBS/van Gieson's stain on MMA embedded tissue. The sections are 5um in thickness and deplasticized. Our protocol seems to work ok on thick sections but not on thin, deplasticized sections. Any thoughts or ideas would be greatly appreciated. Hope you all have a great weekend!! Thanks, Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] harder paraffin
Hello Betsy! I use Leica's EM400 for my paraffin embedding needs and it does extremely well for all of my decalcified bone work. Of course I mostly work with undemineralized bone, biomaterials and medical device implants so I pretty routinely utilize acrylic resins (MMA) for most of my work. With that said, if you need any help or assistance should you need to go the route of plastic embedding, feel free to contact me. One quick note, I also have access to a non-contact laser (laser microtome) that can cut thin micron linear or 3D sections of fresh or resin embedded tissues. If you are interested, I would love to see if this technology could help service your needs if you have some spare tissue samples that you could send over to me. Best Regards, Jack Jack L. RatliffOwner/Histologist - Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for histotechnology317-281-1975jratl...@ratliffhistology.com From: bmolin...@texasheart.org To: Histonet@lists.utsouthwestern.edu Date: Fri, 11 Jan 2013 17:31:02 + CC: Subject: [Histonet] harder paraffin Hi Histonetters, Good Friday to you all. Do you know if there is a “harder” paraffin that may offer more support to the tissue while cutting? I currently use Paraplast Plus and love it, but now I am cutting some polymer material and there is some tearing and was thinking that maybe a different paraffin would help. I am trying to avoid embedding in plastic. Thanks, Betsy Molinari HT (ASCP) Texas Heart Institute Cardiovascular Pathology 1101 Bates Street Houston,TX 77030 832-355-6524 (lab) 832-355-6812 (fax) [THI Celebrates 50 Years]http://www.texasheart.org/AboutUs/History/index.cfm Betsy Molinari Senior Histology Research Technician 832-355-6524 | bmolin...@texasheart.orgmailto:bmolin...@texasheart.org www.texasheart.orgwww.texasheart.orghttp://www.texasheart.org TEXAS HEART INSTITUTE 6770 Bertner Ave. MC 1-283 | Houston, TX 77030 [Receive electronic news from THI]https://secure3.convio.net/thi/site/SPageNavigator/GlobalSiteOptInPage.html[THI on Facebook]http://www.facebook.com/Texas.Heart.Institute[THI on Twitter]http://twitter.com/Texas_Heart[THI on YouTube]http://www.youtube.com/TexasHeartInstitute[THI's Flickr page]http://www.flickr.com/photos/texasheart/sets/ CONFIDENTIALITY NOTICE: The information in this e-mail may be confidential and/or privileged. If you are not the intended recipient, or an authorized representative of the intended recipient, you are hereby notified that any review or dissemination or copying of this e-mail and its attachments, if any, or the information contained herein, is prohibited. If you have received this e-mail in error, please immediately notify the sender by return e-mail and delete this e-mail from your computer system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Maximum bone sample size for methyl methacrylate embedding
Orla, Based upon my personal and professional experience, the sample size of undecalcified bone which can be properly processed into methyl methacrylate for sectioning and staining for Goldner's trichrome is completely dependent upon your microtomy capabilities. For example, if you only have access to a motorized rotary microtome, you are limited to a specimen size that will generally fit onto a 25mm x 75mm microscope slide. In reality, you are looking at a specimen that will be sectioned thin @ approximately 4-6 microns and have a polymerized resin block size no greater than 15mm in width and 20-25mm in cutting length. This means that your specimen needs to fit within these dimensions and at least leave 0.5 - 1.0mm in plastic surrounding the tissue. Again, this is if you are limited by having only a motorized rotary microtome like a Leica RM2255 or RM2265. If you have something like an EXAKT cutting and grinding system for use with cutting thick sections of metallic medical device implants and then polishing sections down to 25-35 microns, you will likely use plastic slides that have a size limitation of 50mm x 100mm. This then means that your resin block will need to be contained within these dimensions of the slide, but without worry to a 0.5mm - 1.0mm plastic resin layer surrounding the tissue because you are cutting thick and grinding/polishing down to a desired final thickness. On the other hand, if you have access to a motorized sledge microtome like a Rechert-Jung Polycut S, E or a Leica SM 2500 for cutting thin 4-6 micron sections, you are only limited by the size of glass microscope slide you can purchase and of course the cutting width of the knife which is 200mm. However, most of my routine work with this piece of equipment is with a resin block limitation of 70mm x 90mm. Please keep in mind that what I am trying to say here is that you are mostly limited by the sectioning capabilities of the equipment that you have access to for microtomy. However, I will caution you that there is also a limitation in size based upon the mastery of the acrylic resin polymerization process. As you go up in size, you increase the difficulty in the ability to manage and control the exothermic polymerization of the specimen and block. Processing and polymerization of larger specimens is absolutely something that can be accomplished without issue, but only by experience that is learned over time through repetition and patience or by training from another that has mastered the technique, something that I have done for others in the past. I hope you understand that working with MMA is not simply something where one can provide you with a protocol and then you can easily expect the same results from one person to another. While I use the same general acrylic resin protocol for every type of bone, I frequently have to adjust the processing and infiltration times to compensate for the size and density of the specimen. I also have to then change how I manage or control the exothermic reaction. This is done by controlling the rate of polymerization, proportionally to the size of the tissue and specimen block, using temperature controls like adjusting room temperature, using a water bath, etc. Now to answer your question, the size you have defined as 2 cm x 1 cm is completely adequate and fairly routine to accomplish. If you message me back privately, I will provide you with a protocol to try. Additionally, if you are doing this for the first time and would like some initial help just to get you past this project until you can practice on your own, I can also offer my services to complete this project for you on contract. Of course, as I stated earlier, I can also train you on how to accomplish this technique as it is also something that I have done in the past for several labs both domestic and internationally. Best Regards, Jack Jack L RatliffRatliff Histology Consultants, LLC317-281-1975 Date: Thu, 13 Dec 2012 17:44:04 + From: o.m.gallag...@sheffield.ac.uk To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Maximum bone sample size for methyl methacrylate embedding Dear Histonetters, Would anyone advise on the maximum size sample of undecalcified bone which could be properly processed into methyl methacrylate for sectioning and staining for Goldner's trichrome? Would anyone have a protocol for processing large bone samples (possibly 2 x1cm) into MMA as most of the protocols I've seen are based on Bordier trephine 5mm iliac crest biopsies. Thank you, Orla -- ** Ms. Orla Gallagher Bone Analysis Laboratory Mellanby Centre for Bone Research D Floor Medical School University of Sheffield Beech Hill Road Sheffield S10 2RX Website: http://mellanbycentre.dept.shef.ac.uk Tel: 00353114-2713337 (office) 00353114-2713174 (lab) E-Mail:o.m.gallag...@sheffield.ac.uk *STOP*: Do
Re: [Histonet] decal affecting IHC
I do not know what is in Calex II but high concentration HCl can have a negative affect. We use 5% formic acid without issue. Jack Jack L Ratliff Owner/Histologist, Ratliff Histology Consultants, LLC Chairman, Hard Tissue Committee - National Society for Histotechnology On Nov 9, 2012, at 11:34 AM, Diana McCaig dmcc...@ckha.on.ca wrote: Does decalcifying tissue (Calex II) affect the antibody reaction for IHC in bony tissue? Diana ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Stain for osteoporotic bone in Spurr plastic
Allison, I am not exactly certain if this protocol will work for Spurr's embedded specimens, but here is the protocol that I use for deplasticized sections of methyl methacrylate embedded undemineralized bone (non-decalcified): Xylenes - warmed @ 50 C for 30 min w/ dip dunk @ 15 min interval Xylenes - warmed @ 50 C for 30 min w/ dip dunk @ 15 min interval Xylenes - warmed @ 50 C for 30 min w/ dip dunk @ 15 min interval 100% EtOH - room temp for 5 min 95% EtOH - room temp for 5 min 70% EtOH - room temp for 5 min DI H2O - room temp for 5 min Silver Nitrate (20 g) + DI H2O (400 ml) - room temp (in dark protected from light) for 5 min DI H2O rinse - room temp (protected from light) for 1 min DI H2O rinse - room temp (protected from light) for 1 min DI H2O rinse - room temp (protected from light) for 1 min Sodium Carbonate (22.5 G) + DI H2O (337.5 ml) + 37% Formaldehyde (112.5 ml) - room temp (protected from light) for 2 min DI H2O rinse - room temp for 1 min DI H2O rinse - room temp for 1 min Sodium Thiosulfate (40 g) + Potassium Ferricyanide (2 g) + DI H2O (420 ml) Solution - room temp for 30 sec(solution stable for 30-60 min after addition of potassium ferricyanide) Running Tap Water Rinse - 10 min Counterstain w/ MacNeal's Tetrachrome (12 g) + DI H2O (600 ml) - room temp for 6-8 min(stir continuously w/ heat @ 60C for 48 hours covered, then filter with paper towel) DI H2O rinse - room temp for 1 min DI H2O rinse - room temp for 1 min DI H2O rinse - room temp for 1 min 70% EtOH - room temp for 1 min 100% EtOH - room temp for 1 min Xylenes - room temp for 3 min Xylenes - room temp for 3 min Coverslip w/ Eukitt Hope this helps! Please feel free to contact me if you have any questions or concerns. Best Regards, Jack Jack L RatliffOwner, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology (317) 281-1975jratl...@ratliffhistology.com LinkedIn Profile: http://www.linkedin.com/profile/edit?trk=hb_tab_pro_top From: allison-malan...@uiowa.edu To: histonet@lists.utsouthwestern.edu Date: Thu, 8 Nov 2012 22:52:42 + Subject: [Histonet] Stain for osteoporotic bone in Spurr plastic Hi there - We are trying to stain osteoporotic bone that was embedded using Spurr plastic. We need to be able to differentiate osteoid from mineralized bone. We have tried Gio's trichrome and Goldner's trichrome with no success, both surface staining and on deplasticized slides. We are thinking of trying Von Kossa, does anyone have a recipe/protocol for this? Or other types of stains that you have had success with? Thank you! Allison Malandra, DVM University of Iowa, Bone Healing Research Lab Notice: This UI Health Care e-mail (including attachments) is covered by the Electronic Communications Privacy Act, 18 U.S.C. 2510-2521, is confidential and may be legally privileged. If you are not the intended recipient, you are hereby notified that any retention, dissemination, distribution, or copying of this communication is strictly prohibited. Please reply to the sender that you have received the message in error, then delete it. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] cutting bone with metal
Thank you Peggy for the kind intro! Both Peggy and Andrew are correct that it is possible to perform histology on this tissue and with the metal remaining intact with the specimen. As they both have stated, resin embedding is required to accomplish this, along with some form of saw sectioning using a diamond studded material like a wire saw, disc wheel, or band saw blade. I have even witnessed and personally used a non-contact laser method to cut sections from specimens containing metallic implants, but that is another discussion in itself! Please feel free to have your researcher contact me directly (see contact info below) Jennifer and I will make sure they get the information they might need to move forward with their study. Best Regards, Jack ratliffj...@gmail.com 615-236-4901 (o) 317-281-1975 (c) On Oct 26, 2012, at 4:16 AM, Lee Peggy Wenk lpw...@sbcglobal.net wrote: Talk with Jack Ratliff, Chair of the NSH Hard Tissue Committee. Jack L. Ratliff 615-236-4901 ratliffj...@gmail.com The answer is Yes, histologic sections can be made, but need plastic resins (methyl methracylate or glycol methacrylate or something similar) and special microtomes and knives. If the researcher's lab doesn't do this technique, Jack can let him know who does, and the tissue can be sent out to the specialty lab. Paraffin blocks on regular histology microtomes won't cut it - literally and figuratively. Peggy Wenk, HTL(ASCP)SLS Beaumont Hospital Royal Oak, MI 48073 The opinions expressed are my own, and do not reflect on Beaumont Hospital. -Original Message- From: Jennifer MacDonald Sent: Thursday, October 25, 2012 11:38 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] cutting bone with metal I have been asked the following question. I do not have an answer and was hoping someone in the Histonet community did. Thanks. There is a researcher who is doing orthopedic procedures on broken rat tibias. The researcher is repairing the tibias with metal rods or plates…not sure which (and the doctor isn't sure what kind of metal either). The researcher wants to know if it is possible to make histologic sections of the repaired tibias with the metal intact ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] histomorphometrics done for bone samples
Hello Hans! If you would like to discuss, please feel free to call me at your convenience. Best Regards, Jack Jack Ratliff Ratliff Histology Consultants, LLC Chairman, Hard Tissue Committee - National Society for Histotechnology On Aug 30, 2012, at 8:37 AM, Hans B Snyder h...@histologistics.com wrote: Hello, I have a Dr. who is asking about how to get histomorphometrics done for bone samples, data needed is % of new bone present in samples from sites grafted with freeze-dried mineralized cancellous allograft bone. We can do the plastics on it but the computer assisted part, I do not know anything about. Thanks Hans B Snyder Histologistics 100 Barber ave Worcester, MA 01606 www.histologistics.com h...@histologistics.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] National Society for Histotechnology Hard Tissue Forum Event - Bethesda, MD on August 18, 2012!
The Hard Tissue Committee of the National Society for Histotechnology would like to remind everyone that we are hosting a singe day Hard Tissue Forum event at the Doubletree by Hiltion in Bethesda, MD on August 18th! You wont want to miss out on this opportunity to learn about and witness first hand all current forms of resin/plastics histology, as well as see for the first time ever here in North America a newly developed non-contact laser microtome! 7:30am - Registration Opens 8:00am – Hard Tissue Histology: An Historical And Current Perspective of Microtomy Technique – Jack L Ratliff 9:00am – Skeletal Analysis With MicroCT: What You Need To Know And Why You Need To Know It – Daniel S Perrien, Ph.D. 10:30am – Refreshment Break 10:45am – Bone, Biomaterials And Bugs: Resin Microtomy And The Rotary Microtome – Damien Laudier, BS, HTL(ASCP), QIHC 12:15pm – Lunch On Your Own 1:15pm – Ground Section Microtomy Techniques (Parts I II): Diamond Materials Combined With Manual And Semi-Automated Grinding Methods To Collect And Polish A Variety Of Resin Embedded Specimens – (PART I) Joe Tabeling Jack L Ratliff, BA; (PART II) Linda Durbin Robert A Skinner 2:45pm – Refreshment Break 3:00pm – New Results In Laser Sectioning For General Histology, Tissue Engineering, Medical Device Implants And Industrial Analyses – Heiko Richter, Ph.D. 4:00pm – Histomorphometry of Bone: A Quantitative Description of Bone Histology Using Sub-Micron Resolution Optical Microscopy – Nathanael H Reveal You can still register online at https://s3.goeshow.com/nsh/2012HT/ereg572259.cfm?pg=register or show up at the program and register onsite at 7:30am before the start of the meeting. We hope to see you all there on the 18th! Best Regards, Jack RatliffChairman, Hard Tissue Committee - National Society for Histotechnology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] National Society for Histotechnology Hard Tissue Forum Event - Bethesda, MD on August 18, 2012!!!
Greetings! The Hard Tissue Committee of the National Society for Histotechnology would like to remind everyone that we are hosting a singe day Hard Tissue Forum event at the Doubletree by Hiltion in Bethesda, MD on August 18th! You won't want to miss out on this opportunity to learn about and witness via LIVE demonstration all current forms of resin/plastics histology equipment. A special treat this year will be a first ever showing and demonstration in North America of a newly developed non-contact laser microtome (TissueSurgeon) to cut micron thin sections of a variety of tissue types! 7:30am - Registration Opens 8:00am – Hard Tissue Histology: An Historical And Current Perspective of Microtomy Technique – Jack L Ratliff 9:00am – Skeletal Analysis With MicroCT: What You Need To Know And Why You Need To Know It – Daniel S Perrien, Ph.D. 10:30am – Refreshment Break 10:45am – Bone, Biomaterials And Bugs: Resin Microtomy And The Rotary Microtome – Damien Laudier, BS, HTL(ASCP), QIHC 12:15pm – Lunch On Your Own 1:15pm – Ground Section Microtomy Techniques (Parts I II): Diamond Materials Combined With Manual And Semi-Automated Grinding Methods To Collect And Polish A Variety Of Resin Embedded Specimens – (PART I) Joe Tabeling Jack L Ratliff, BA; (PART II) Linda Durbin Robert A Skinner 2:45pm – Refreshment Break 3:00pm – New Results In Laser Sectioning For General Histology, Tissue Engineering, Medical Device Implants And Industrial Analyses – Heiko Richter, Ph.D. 4:00pm – Histomorphometry of Bone: A Quantitative Description of Bone Histology Using Sub-Micron Resolution Optical Microscopy – Nathanael H Reveal You can still register online at https://s3.goeshow.com/nsh/2012HT/ereg572259.cfm?pg=register or show up at the program and register onsite at 7:30am before the start of the meeting. We hope to see you all there on the 18th! Best Regards, Jack Ratliff Chairman, Hard Tissue Committee - National Society for Histotechnology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] NATIONAL SOCIETY FOR HISTOTECHNOLOGY - HARD TISSUE FORUM EVENT AUGUST 18th in BETHESDA, MD!!!!!!
The Hard Tissue Committee of the National Society for Histotechnology would like to remind everyone that we are hosting a singe day Hard Tissue Forum event at the Doubletree by Hiltion in Bethesda, MD on August 18th! You wont want to miss out on this opportunity to learn about and witness first hand all current forms of resin/plastics histology, as well as see for the first time ever here in North America a newly developed non-contact laser microtome! 7:30am - Registration Opens8:00am – Hard Tissue Histology: An Historical And Current Perspective of Microtomy Technique – Jack L Ratliff9:00am – Skeletal Analysis With MicroCT: What You Need To Know And Why You Need To Know It – Daniel S Perrien, Ph.D.10:30am – Refreshment Break10:45am – Bone, Biomaterials And Bugs: Resin Microtomy And The Rotary Microtome – Damien Laudier, BS, HTL(ASCP), QIHC12:15pm – Lunch On Your Own1:15pm – Ground Section Microtomy Techniques (Parts I II): Diamond Materials Combined With Manual And Semi-Automated Grinding Methods To Collect And Polish A Variety Of Resin Embedded Specimens – (PART I) Joe Tabeling Jack L Ratliff, BA; (PART II) Linda Durbin Robert A Skinner2:45pm – Refreshment Break3:00pm – New Results In Laser Sectioning For General Histology, Tissue Engineering, Medical Device Implants And Industrial Analyses – Heiko Richter, Ph.D.4:00pm – Histomorphometry of Bone: A Quantitative Description of Bone Histology Using Sub-Micron Resolution Optical Microscopy – Nathanael H Reveal You can still register online at https://s3.goeshow.com/nsh/2012HT/ereg572259.cfm?pg=register or show up at the program and register onsite at 7:30am before the start of the meeting. We hope to see you all there on the 18th! Best Regards, Jack RatliffChairman, Hard Tissue Committee - National Society for Histotechnology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] 2012 Hard Tissue Forum Event in Bethesda, MD (August 18th!)
Hello! My name is Jack Ratliff and I am Chairman of the Hard Tissue Committee (HTC) for the National Society for Histotechnology (NSH). On behalf of the HTC and the NSH, we would like to invite you to attend and participate in the 2012 Hard Tissue Forum. This event will take place August 18th, 2012 and will be held at the Doubletree by Hilton in Bethesda, MD. We have put together an exciting program this year and we invite you to be a part of it! The program this year will focus on relevant histological and image analysis techniques associated with undemineralized bone and medical device implants. While the topics of microtomy (thin ground sectioning) and image analysis (microCT histomorphometry) will be highlighted, the sub-category of workshops will showcase a concentration of equipment, techniques, and technology that promises to be a unique single-day event demonstrating a hands-on application of specific techniques never before staged together in a single histology event! In fact, one of the highlights of this years program will be a first ever in the U.S and North America unveiling and demonstration of a non-contact laser microtome that has been developed to take thin sections of fresh and resin/plastic embedded tissues! To learn more about the Hard Tissue Forum, please visit http://s3.goeshow.com/nsh/2012HT/ereg572259.cfm?clear where you can find speaker related information, detailed workshop abstracts, and event registration information. If you have ANY questions, please feel free to contact Aubrey Wanner, Meeting Manager of the NSH (aub...@nsh.org) or myself (ratliffj...@hotmail.com) at your convenience. I look forward to seeing you in Bethesda! Yours Sincerely, Jack Jack L RatliffChairman, Hard Tissue Committee - National Society for Histotechnology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Staing rack for ground section??
Marc, May I ask what staining protocol you are using, typical section thickness and what type of specimens you are staining? The problem is that no such product exists and most likely because some believe that you cannot batch stain 50 x 100mm EXAKT slides due to the complexity of some staining protocols, tissue types, and lack of true consistency from a section thickness perspective. I could help steer you in the right direction, but I will need to know more about what you are working with in order to provide the best advice. Best Regards, Jack Jack Ratliff Chairman, Hard Tissue Committee - National Society for Histotechnology Date: Thu, 28 Jun 2012 10:36:16 -0500 From: boneima...@gmail.com To: Histonet@lists.utsouthwestern.edu CC: Subject: [Histonet] Staing rack for ground section?? Hello Histoland! I've recently been tasked with doing some ground section work and can not find a staining rack suited specifically for 50 x 100mm slides. Does anybody know where I can find one? Thanks, Marc DeCarlo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Re: Staing rack for ground section??
Marc, My apologies for butting in here, but basically the length of the rack is a bit larger than the 100 mm slides and a looser fit. Also, the height of the slides is larger than the rack inserts and can sometimes create a stability problem when staining a full rack. As Andrew has stated, it is the next best thing and this product can work for you. However, the staining dish is also a problem because the slides do not stain vertical in the traditional sense meaning that you need the larger 600 ml volume dishes. If you would like to contact me privately, I would love to speak with you more about what you are trying to accomplish and help point you in the right direction with this sort of thing. Best Regards, Jack Date: Thu, 28 Jun 2012 15:54:27 -0500 From: boneima...@gmail.com To: petticoffer.and...@synthes.com CC: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: Staing rack for ground section?? Thanks for all the advice and help from those that responded. Your knowledge is greatly appreciated. @ Andy, you mentioned the slides don't quite fit; is it the length or height that doesn't fit? Thanks in advance, Marc D On Thursday, June 28, 2012, petticoffer.and...@synthes.com wrote: Sorry the link got cut off. To get to the correct slide rack, search catalog #3004 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu javascript:; [mailto:histonet-boun...@lists.utsouthwestern.edu javascript:;] On Behalf Of petticoffer.and...@synthes.com javascript:; Sent: Thursday, June 28, 2012 1:13 PM To: boneima...@gmail.com javascript:;; Histonet@lists.utsouthwestern.edujavascript:; Subject: RE: [Histonet] Staing rack for ground section?? Check out the link below for BRL. The slides aren't a perfect fit but will stay in place if you're careful. I usually space the slides every other in case of any movement, but have done more with good results. It's the best things I've found to date for that size slide and has been a real time saver. http://brainresearchlab.com/online-catalog/steel-staining-rack-30-slides -4/ Andy Andrew Petticoffer Research Associate DePuy Synthes - Biomaterials 1230 Wilson Drive West Chester, PA 19380 Phone: (484) 356-9576 Fax: (484) 356-9591 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu javascript:; [mailto:histonet-boun...@lists.utsouthwestern.edu javascript:;] On Behalf Of Marc DeCarlo Sent: Thursday, June 28, 2012 11:36 AM To: Histonet@lists.utsouthwestern.edu javascript:; Subject: [Histonet] Staing rack for ground section?? Hello Histoland! I've recently been tasked with doing some ground section work and can not find a staining rack suited specifically for 50 x 100mm slides. Does anybody know where I can find one? Thanks, Marc DeCarlo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu javascript:; http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu javascript:; http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] blocks processed in plastic
Hello Nancy! What type of plastic blocks are you wanting to cut? May I also ask what type of specimens are contained within the blocks. Without much information on what specifically you are working with you are looking at needing diamond knives, tungsten-carbide knives, or diamond impregnated wire, band saw blades, or disc wheels. Best, Jack Jack Ratliff Senior Histologist, BioMimetic Therapeutics, Inc. Chairman, Hard Tissue Committee - National Society for Histotechnology From: nancy_schm...@pa-ucl.com To: histonet@lists.utsouthwestern.edu Date: Mon, 30 Apr 2012 17:04:22 + Subject: [Histonet] blocks processed in plastic Hi Histonetters- Is there a special blade or angle or directions required for cutting plastic blocks? As always - Thanks for your help Nancy NOTICE: This email may contain legally privileged information. The information is for the use of only the intended recipient(s) even if addressed incorrectly. If you are not the intended recipient, please notify the sender that you have received it in error and then delete it along with any attachments. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Diamond Knives
Have a look at Delaware Diamond Knives (DDK). Their website I believe is www.ddk.com. I'm sure they can help you or at a minimum point you in the right direction. Best Regards, Jack On Mar 14, 2012, at 9:42 PM, Philip Slakmon slak...@yahoo.com wrote: Good Afternoon, I was interested in knowing people's opinions on the different diamond knives on the market. Opinions could be based on quality, design, workmanship, delivery, customer service/support, pricing, Thank you, Philip ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Processor Preferences??
John, Go for the ASP300S and then let's talk again at your convenience about resin infiltration needs. I hope the information I sent to you was both informative and helpful! Regards, Jack On Mar 15, 2012, at 9:13 AM, John Baker bak...@umich.edu wrote: Hello Jennifer, I saw your message about tissue processors on the Histonet Archive May 2006 and wondered which unit you decided to purchase? We are looking for one now and have three in mind, Thermo Pathcentre, Leica ASP300s and the Tissue-Tek VIP6. Your thoughts on your choice and on these listed. Thanks you, John John A. Baker The University of Michigan Orthopaedic Research Laboratories Histology Unit 109 Zina Pitcher Place, 2218 BSRB Ann Arbor, MI 48109-2200 734-936-1635 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Diamond Knives
Philip, Marc is correct in everything that he has stated, but if you are looking for diamond knife purchases or services as you indicated in your original posting, I am only aware of DDK providing this service. Best Regards, Jack Jack Ratliff Hard Tissue Histologist Chairman, Hard Tissue Committee - National Society for Histotechnology On Mar 15, 2012, at 9:10 AM, Marc DeCarlo boneima...@gmail.com wrote: Philip, We also use Dorn and Hart Microedge. As far as I know they only do Tungsten Carbide and steel knives but if that is what your looking for they are the best priced, quality, and easiest to work with. Marc On Thursday, March 15, 2012, Bernice Frederick b-freder...@northwestern.edu wrote: We have a DDK knife, works great on undecalcified bone. We send it to Dorn and Hart Microedge for resharpening as they are local to us and have been at it for years. They used to sharpen our old microtome knives. Talk about dating yourself! Bernice Bernice Frederick HTL (ASCP) Senior Research Tech Pathology Core Facility ECOGPCO-RL Robert. H. Lurie Cancer Center Northwestern University 710 N Fairbanks Court Olson 8-421 Chicago,IL 60611 312-503-3723 b-freder...@northwestern.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jack Ratliff Sent: Thursday, March 15, 2012 8:20 AM To: Philip Slakmon Cc: Histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Diamond Knives Have a look at Delaware Diamond Knives (DDK). Their website I believe is www.ddk.com. I'm sure they can help you or at a minimum point you in the right direction. Best Regards, Jack On Mar 14, 2012, at 9:42 PM, Philip Slakmon slak...@yahoo.com wrote: Good Afternoon, I was interested in knowing people's opinions on the different diamond knives on the market. Opinions could be based on quality, design, workmanship, delivery, customer service/support, pricing, Thank you, Philip ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Re: undecalcified bone IHC
I might also add that Neil Hand is co-speaking with myself and Philip Seifert this year at the annual National Society for Histotechnology - Symposium/Convention in Vancouver B.C. Our workshop is titled: Resin Applications Forum: Methods for Processing, Special Staining, Immunohistochemical and In Situ Hybridization of Soft and Hard Tissue Including Medical Device Implants During the last 50 years, numerous histological procedures have been described on resin embedded tissue. While different types of resins are available for different purposes, the acrylics provide the widest range of techniques, especially for light microscopy applications. However, as demand from HE to more sophisticated techniques increases, so too have the problems, and nowhere is this more apparent and controversial than in the application of immunohistochemistry on resin sections. This workshop will provide a review and discussion for those individuals that currently work with and/or are just getting started working with soft and hard tissue specimens and specifically the various resins (i.e. MMA, GMA, Technovit, Acrylosin, etc.) associated with their specific tissue interests. The workshop will also detail the preparation and staining of sections of soft and hard tissue, including implants (e.g. undemineralized bone and cardiovascular stents), for immunohistochemical and in situ hybridization staining using different acrylic and epoxy resin embedding media. Specific problems and pitfalls, either technical or operational associated with certain resin embedding procedures, will be illustrated and examined. Particular emphasis will be given to procedures which have been used extensively for routine diagnostic, and research purposes, i.e. those that WORK! Individuals with a current or future intent to process and cut undemineralized tissue or tissue containing foreign implant materials using acrylic or epoxy resins are strongly encouraged to attend this workshop! Please feel free to contact me if you would like more information about the workshop as information relevant to the exact date and time becomes available. All I know at this time is that the NSH meeting is September 29th - October 3rd, 2012. Best Regards, Jack Jack Ratliff Hard Tissue Histologist Chairman, Hard Tissue Committee - National Society for Histotechnology From: gayle.cal...@bresnan.net To: histonet@lists.utsouthwestern.edu Date: Mon, 12 Mar 2012 11:04:20 -0600 Subject: [Histonet] Re: undecalcified bone IHC Jeff, It is most certainly possible to do IHC on undecalcifed bone sections embedded in PMMA although not the easiest task. Sectioning is done on a microtome that is powerful enough to cut the plastic and using tungsten carbide knives. The key is total removal of the plastic from MMA embedded bone sections to allow antibody/ immunoglobulins to access antigenic sites. Neil Hand has done IHC successfully on PMMA embedded tissues including undecalcified bone on 2 to 3 µm thick sections. I think one could cut thicker sections at 4 to 5 µm and still be successful. I do not recall what Troiano et al used. The following publications will help you and should include protocols, although conventional protocols will work according to Hand. Blythe D. Hand N et al 1997 J Clin Path 50:45-49. The use of methyl methacrylate resin for embedding bone marrow trephine biopsies. Hand NM et al 1996 Antigen unmasking using microwave heating on formalin fixed tissue embedded in methyl methacrylate J Cellular Path 1:31-37 Jackson P et al. 1996 Amplification of immunocytochemical reactions by the catalytic deposition of biotin on tissue sections. J Path 170(suppl):23A. This was about tyramide amplification when one gets a weak signal from conventional methods. Hand NM, Church RJ 1998 Superheating using pressure cooking: its use and application in unmasking antigens embedded in methyl methacrylate. J Histotechnology 2`:231-236 Hand NM et al 1989 Immunohistochemistry on resin embedded tissue for light microscopy: a novel post embedding procedure. Proceeding Royal Microscopical Society 24(1):A54-55. Hand NM Plastic Embedding media and techniques, Ch.30, p 663-677. Theory and Practice of Histological Technique, 5th edition by Gamble and Bancroft. The 6th edition is updated under same title. Use Google Scholar to find Troiano N et al from Yale on doing IHC on PMMA embedded bone sections with publications in J Histotechnology. Hand mentioned several HIER methods, using citrate buffer. Optimizing retrieval will depend on the antigen and you may end up doing this with some form of HIER, including microwave or other heat producing methods and with different buffers. Enzyme digestion is also a possibility. Hand removed MMA with xylene, warm my speed up the removal, also more than one change for 10 - 20 minutes or longer. When I talked to him
RE: [Histonet] Goldner's Trichrome for Osteoid
Andi, Your PI has requested the Goldner's trichrome stain because it provides a stark contrast between the green (light-green SF yellowish) stained mineralized component of bone and the red (acid fuchsin-ponceau) stained newly formed unmineralized bone-like dense collagen fibers (osteoid) that will eventually mature and mineralize to form bone. Now if you subject mineralized bone to an acid solution, the process of demineralization begins and acts to remove the mineral (calcium and phosphorus) content. The acid will also have an effect on the soft tissue components, but the effect varies depending upon the acid concentration and certain properties of tissues. Nevertheless, the most notable effect of acid decalcification will be seen as a change in the density of bone and demonstrated by stain uptake and penetration. If you have to demineralize your bone in order to cut sections because you do not have the capabilities to cut undemineralized bone, then you need a stain that provides the best contrast for this now demineralized state of bone. Once the bone has been decalcified, you essentially have now demineralized bone (less dense) and still unmineralized bone-like collagen fibers, all of which will now be contrasted differently with the Goldner's stain. While an HE can be used to distinguish bone that was once mineralized (old bone) from unmineralized bone-like collagen fibers (osteoid or newly forming bone), there is not enough of a stark contrast and especially if you are wanting to use some form of image analysis software. With that said, the best stain then to use in this situation is a Masson's trichrome where the bone stains blue (aniline blue) and the osteoid stains red (biebrich-scarlet). With regards to the demineralization method, the EDTA will take too long to arrive at the same result that a simple 5% formic acid will provide. Also, your sections will be cut at a standard 5 microns. Hope this information helps you to get what you need and understand a little more of why you need what you need! :) Best Regards, Jack From: algra...@email.arizona.edu To: histonet@lists.utsouthwestern.edu Date: Thu, 8 Mar 2012 08:00:58 -0800 Subject: [Histonet] Goldner's Trichrome for Osteoid Hopefully a hard tissue guru is out there and can help with this. A PI here is asking about doing a Goldner's stain on mouse femur. I'm not set up to cut sections of undecalcified bone so my questions are: 1. can this stain be done successfully on decalcified bone? 2. if so, what type of decal should be used - EDTA? 3. I have googled the stain but didn't see what the preferred thickness was for this stain. Need this info fairly soon! The bones are on the way. Thanks!!! Andi G. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Optimizing HE Stains for undecalcified bone
Merissa, You should very easily be able to get a nice looking HE from deplasticized undemineralized bone sections. In fact, try the protocol that I routinely use for my MMA embedded specimens: Deplastification x 3 changes each for 15-30 minutes @ 55-60C w/ gentle dip and dunk at half way mark for each change 100% EtOH @ RT for 5 min 95% EtOH @ RT for 5 min 70% EtOH @ RT for 5 min dH2O @ RT for 5 min Hematoxylin 2 (from Richard Allen @ Fisher Sci) @ RT for 3 min Running Tap Water Rinse for 1 min Clarifier 2 (from Richard Allen @ Fisher Sci) @ RT for 20-30 seconds with a gentle dip and dunk at half way mark Running Tap Water Rinse for 1 min Bluing Solution (from Richard Allen @ Fisher Sci) @ RT for 20-30 seconds with a gentle dip and dunk at half way mark Running Tap Water Rinse for 1 min Eosin Y (from Richard Allen @ Fisher Sci) @ RT for 40 seconds 70% EtOH @ RT with 10 gentle dip and dunks 95% EtOH @ RT with 10 gentle dip and dunks 100% EtOH @ RT with 10 gentle dip and dunks 100% EtOH @ RT for 30 seconds Xylenes @ RT for 3 min Xylenes @ RT for 3 min Coverslip Your problem could be the type of hematoxylin you are using (progressive vs. regressive) and the length of time in solution. I would also recommend you try a Von Kossa with a MacNeal's tetrachrome counterstain, Goldner's trichrome, and maybe a toluidine blue stain with these type of sections. Best Regards, Jack Jack Ratliff Chairman, Hard Tissue Committee - National Society for Histotechnology On Mar 8, 2012, at 3:51 PM, M.O. modz9...@gmail.com wrote: Hello Histonetters! I am new to staining and can't seem to get good HE stains from deplacticized undecalcified bone with an implant. It seems as though the hematoxylin is staining too dark. Any feedback would be much appreciated! The protocol I am using is for after the deplasticization step and says: 100% EtOH for 3 mins, 70% EtOH for 3 mins Hematoxylin for 6mins, Rinse, .25% Acid Ethanol dip 8-10 times, Rinse 95% EtOH 1min Eosin for 2mins Quick Dip 95%, 95%, 100% 100% EtOH Soak in ProPar for 15+mins Sincerely, Merissa ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC and bone implants
Del, I have a few questions for you to better understand your problem. 1) Are you talking about decalcified paraffin sections or undecalcified resin embedded specimens? 2) Is the implant washing off or both the bone and implant? 3) What type of implant/material is it? 4) If decalcified and paraffin embedded, what temperature and how long do you melt your specimens to the slide on the slide warmer? 5) If undecalcified and resin embedded, what type of resin and how thick are your sections? 6) Lastly, what step do you notice that the section or implant becomes detached from the slide? Please forgive me for the list of questions, but a little more information will help to pinpoint the problem and help get straight to the best corrective action. Best Regards, Jack Jack Ratliff Chairman, Hard Tissue Committee - National Society for Histotechnology Senior Histologist, Biomimetic Therapeutics, Inc. From: dphill...@vetmed.lsu.edu To: Histonet@lists.utsouthwestern.edu Date: Mon, 13 Feb 2012 09:53:15 -0600 CC: Subject: [Histonet] IHC and bone implants Does anyone have any suggestions for staining bone implants? I am having trouble with wash offs. I use charged slides and they are still washing off. Thanks Del ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Hard tissue microtome
Can you tell us more about the bone specimens you are wanting to cut? Jack Jack Ratliff Senior Histologist, BioMimetic Therapeutics, Inc. Chairman, Hard Tissue Committee - National Society for Histotechnology Date: Thu, 22 Dec 2011 10:25:35 -0800 From: silvinamolinu...@yahoo.com.ar To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Hard tissue microtome Hi! could anyone recommend me a good microtome for hard tissue (specifically for bone tissue)? Thanks' in advance Hope you all have a good nativity and happy new year! silvina ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Formic Acid Recipe
Hello Jesus! Try Sanderson's Rapid Bone Stain from Dorn and Hart Microedge (www.dornandhart.com). This is a metachromatic stain and should help you to penetrate the resin if heated at 60C and stained for 5-10 minutes depending upon section thickness. Rinse the slide in dH2O @ 60C and blot dry gently. Stain longer or shorter as necessary. Jack From: jesus.w@gmail.com Date: Fri, 4 Nov 2011 16:41:20 -0500 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formic Acid Recipe Dear all, I was having trouble staining Human embryonic palatal mesenchymal cells with multi-stain solution. My PI told me to use formic acid on the samples so that it could increase the permeability of the methyl methacrylate. I am not sure if this is enough information, but the cells were loaded on hydroxyapatite scaffolds. I have also tried aniline blue and villaneuva stain. Any information on how I can make a solution of formic acid is appreciated along with maybe other stains I could try using. Each sample was grinded to about 50 microns. Thank you. Best Regards, Jesus W. Hernandez Graduate Student Department of Biomedical Engineering University of Texas at San Antonio jesus.w@gmail.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Technovit 9100 New
What is it you are trying to accomplish with staining? Can you tell us more about your project? I use Acrylosin SOFT Infiltration and Embedding solutions from Dorn and Hart Microedge (just up the road from you in Villa Park) for my thin 5 micron section and Acrylosin HARD for ground sections. Feel free to ask more questions as needed. Jack Senior Histologist, BioMimetic Therapeutics Chairman, Hard Tissue Committee - National Society for Histotechnology On Oct 16, 2011, at 10:47 PM, Megan Silas sil...@illinois.edu wrote: Hello, Has anyone used Technovit 9100 New? If so, what company did you order through - is there a distributor in the US? Also, have you found success with this in embedding undecalcified bone? Thanks for any and all advice! Megan Silas Megan Silas | megan.r.si...@gmail.com | 847-609-6336 University of Illinois at Urbana-Champaign | Bioengineering ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Von Kossa stain kit
It has been my experience that you do not need to acid wash the glassware for the silver nitrate step. The only critical parts are the time (5 minutes) in silver nitrate (protected from light), the 3 dH2O rinses (also protected from light), developing time (2 minutes) in sodium carbonate-formaldehyde solution (protected from light), 2 dH2O rinses (no need to protect from light), the 30 seconds in Farmer's Diminisher (sodium thiosulfate-potassium ferricyanide solution) and tap water rinse. The sodium-carbonate formaldehyde solution is use to chemically develop the silver attachment to any presence of mineralization. Most people use natural light, but the chemical step is more consistent in my opinion. Additionally, since the silver nitrate step is used to attach silver ions to the presence of minerals, the use of any metal is prohibited as it will also bind silver. However, if you use plastic, glass, or even stainless steel, no worries at all. As for a reliable kit, I use the kit from Dorn and Hart Microedge (www.dornandhart.com). You can also choose you preferred counterstain with this kit. I routinely work with mineralized bone and therefore use the Von Kossa - MacNeal's Tetrachrome stain kit. Hope this helps! Best, Jack Date: Tue, 4 Oct 2011 11:02:45 -0400 From: msherw...@partners.org To: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Von Kossa stain kit Thought of another question! What other vendors do people order special stain kits? We have been using Poly Scientific, which is very good, but costly. Thanks! Peggy Peggy Sherwood Lab Associate, Photopathology Wellman Center for Photomedicine (EDR 214) Massachusetts General Hospital 50 Blossom Street Boston, MA 02114-2696 617-724-4839 (voice mail) 617-726-6983 (lab) 617-726-1206 (fax) msherw...@partners.org The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] iliac artery attachment to slides?
Jim, I think that I might better understand you now from your reply and would like to share my response with others that might be following this thread. From the looks of it you are using 50% EtOH on the slide before rolling out the section. You then mentioned that you once used a drop of xylenes instead of the 50% EtOH and everything works, but you lose sections during deplastification. My routine procedure is to first lubricate the block during cutting with 70% EtOH, I then use 95% EtOH on the slide and periodically (if needed) on section during the process of rolling out the section and orientating on the slide, with the section wet with 95% EtOH I place a section cover over the top, then using bibulous paper and a brayer roller I gently start in the middle rocking back and forth while gradually pressing down with more force until I have attached the section to the slide. This typically help to eliminate wrinkles, however, with your type of specimens you can still have trouble so you need to improvise with the actually pressing method where you actually attach the section to the slide. If you have not tried the higher concentrations of ethanol during the roll out steps, try it and let me know your results. Please keep in mind that I am assuming that you are using something like Haupt's Adhesive (Dorn and Hart Microedge) to coat your slides and that your resin blocks are fairly soft. You see, the higher alcohol concentration acts to help soften the resin so that it is easy to unroll the section and eliminate wrinkles. In fact, the concentration of ethanol and its effectiveness during this step is proportional to the hardness or softness of the resin block. I know that you use MMA + DBP + P-16, but can you tell us the concentrations? I use Acrylosin Infiltration Embedding solution for my resin work now and the SOFT product contains 10% of the softening component. One other thing to try with the attachment of these difficult type specimens is to float them out on the slide in a pool of 95% EtOH on a slide warmer. The 95% will help you to tease out the wrinkles and the heat of the slide warmer will help to evaporate the ethanol so that you can get some sort of initial attachment to the Haupt's before you try to press the section flat. Also, before you place the section cover on top of the section, add a drop of further diluted Haupt's to the section so that it can dry with the section just in case you are worried about gelatin coating loss. You might get some additional background, but at least it is something that you can tweak if the ethanol steps help to increase your ability to eliminate wrinkles. Jack Date: Wed, 31 Aug 2011 09:04:28 -0500 From: herrick.ja...@mayo.edu To: histonet@lists.utsouthwestern.edu Subject: FW: [Histonet] iliac artery attachment to slides? Good morning everybody!! I am trying to attach iliac arteries embedded in MMA (methyl methacrylate + dibutyl phthalate + perkadox 16) to gelatin coated slides to allow us to stain them with the Movat's Pentachrome. Unfortunately, I have a real problem getting them to attach - shortly after they are placed into 2-MEA for plastic removal, the section detaches from the slide. I have mounted hundreds of sections to slides before with great success, but have never tried iliac arteries before. When mounting them using the conventional method (a drop of 50% ETOH, roll them onto the slide using a plastic coverslip, clamping the stack and placing it into a 45 - 50 degree Celcius oven for 24 to 48 hours), the sections attach beautifully and do not fall off during deplasticization. The problem with the iliac sections is that they are completely consumed with wrinkles around the entire circular area of the artery, rendering them unuseable. I have tried dry mounting (without using 50% ETOH or dH2O), I have tried floating the section onto a slide from a warm ETOH or dH2O solution (in water bath), I have tried using a slide warmer, I have tried a heat gun at low and high temperature settings, etc., etc.. The sections look great using the dry mounting methods (i.e. they don't wrinkle), but they detach from the slide very quickly following submersion in 2-MEA. It seems to me that the wrinkles appear, following the introduction of the ETOH or dH2O. If anyone would have any suggestions or comments, it would be greatly appreciated. Thanks again for all of your help. Have a great day!! Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu
RE: [Histonet] Re: iliac artery attachment to slides?
Jim, I agree with Teri regarding the Haupt's Adhesive and assume that when you mean a gelatin coating that it is possible that you might already be using this product based upon past conversations. If you are using Haupt's, then I am confused that you are having difficulty with your sections remaining attached to the slide. I am in that group Teri mentioned that swear by Haupt's and it ability to retain sections throughout staining. This then leaves me to question the use of 2-MEA for removal of the resin since you seem to be having problems after this step. Please remember a few things (my opinion and I welcome the challenge from others) when mounting resin sections to gelatin coated slides: #1 - The concentration of gelatin in the coating solution is directly proportional to the adhesive property of the section to the slide. #2 - The concentration of the gelatin in the coating solution is directly proportional to the background staining of certain high molecular weight dyes (i.e. hematoxylin, Aniline blue, etc.). #3 - The adhesive property of the gelatin, attachment of the section to the slide, is proportional to the heat activation of the gelatin during the slide section drying phase (i.e. heating in an oven for a period of time at a certain temperature range). Allow me to briefly expand upon this last statement. The best way that I can explain all of this to people is what I simply call the tape effect. If you use regular Scotch tape on paper in a room temperature environment, 8 or 9 times out of ten you can safely remove the tape without tearing the paper and without a sticky residue. If you repeat the same procedure in a warmer environment, like say you left it on the paper and stuck it to a window inside your car with all the windows up on a hot summer day, you will be able to remove the tape while it is hot without damage to the paper, but you will leave a very sticky residue on both the paper and the window due to the heat activation of the adhesive. However, if you wait until late evening to perform the same task after the tape has cooled, the adhesion is much stronger and you are highly likely to tear the paper and find it difficult to remove easily from the window. This is how I explain heat activation of the gelatin adhesive and it is for this reason that I use an aluminum slide press as a heat sink to help dry ALL slide sections evenly and activate the adhesive properties of the gelatin by increasing the internal heat within the slide chambers. Essentially, you are looking for the correct proportion of adhesive concentration to background staining, all while avoiding the melting of the plastic section covers but still so that all slides dry evenly and so that enough heat is achieved to activate and optimize the adhesive properties of the gelatin (Haupt's Adhesive) coated slides. I then deplastify in warmed xylenes at 50 C, three fresh changes for 30-60 minutes depending upon the overall size of the sections (i.e. small sections on 1x3 slides or large sections on 2x3 slides) and do not lose sections! One last thing to consider is that the geometry of your tissue (circular) is also a contributing factor because of the shrinking and swelling effect in using organic solvents like xylenes to deplastify, ethanol's of decreasing concentration to hydrate the sections, water based solutions for staining and then dehydrating again to coverslip. The density of the tissue then also increases this shrinking and swelling effect. For example, this is not as noticeable with undemineralized bone. So, what do you do? Well, obviously you have to identify where your weaknesses are and then start changing the variables one-by-one until you optimize the method. If it was me I would eliminate the 2-MEA first, then ensure adequate and even drying of the slides as also related to adhesive activation in the oven. If you are still having problems, play with the concentration of the gelatin adhesive and learn to live with the background. There really is a lot of directions to go here, but by no means throw in the towel and give up! Hope I have been helpful and not too confusing. :) Best, Jack Simple concept I know, but think about this, the proper balance of gelatin concentration, heating and cooling during the drying phase, subsequent use of chemicals, and the density properties of the tissue, all contribute to the overall section adhesion throughout staining. From: t...@stowers.org To: histonet@lists.utsouthwestern.edu Date: Mon, 29 Aug 2011 13:18:32 -0500 Subject: [Histonet] Re: iliac artery attachment to slides? Hi Jim, sounds like you are having a time of it. I figure Jack Ratliff will chime in as soon as he sees this. In the meantime I will give you the same advice he gave to me. If you are having troubles with tissues adhering, try Haupt's adhesive. You can find recipes on the internet to make
RE: [Histonet] heavy duty microtome
I know of two possible options for exactly what you seek. Please contact me directly so that we may point you in the right direction and discuss with you the details. Best Regards, Jack PS I have heard a rumor that these units will no longer be for sale by Leica and no replacements will be available in the future. Additionally, I very seriously doubt that they have a demo unit for sale as they typically manufacture these units upon approved purchase order. Jack L Ratliff Senior Histologist, BioMimetic Therapeutics, Inc. Chair, Hard Tissue Committee - National Society for Histotechnology From: j...@ana.au.dk To: histonet@lists.utsouthwestern.edu Date: Fri, 26 Aug 2011 09:08:29 +0200 Subject: [Histonet] heavy duty microtome Does anyone have either a used Leica Polycut E or SM 2500 for sale. For the present we are using a very old (50 years) Reichert-Jung heavy duty microtome and looking for a replacement. If you can help us please contact: Jette Barlach Research unit for Rheumatology an Bone biology Dept. of Rheumatology University hospital of Aarhus Nørrebrogade 44 8000 Aarhus C Denmark j...@ana.au.dkmailto:j...@ana.au.dk +45 89 42 29 92 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] cadaveric bone
Louise, My first suggestion to you is to de-wax the specimen and process into an MMA based resin containing a softener like DBP. Once polymerized you can simply take thin sections using tungsten-carbide knives. Make sure to infiltrate the specimen well with the resin solution prior to embedding. If you are unable to try this method, I would be happy to try this for you free of charge if you could send me the specimen. Regarding further specimens of this type, again just process, infiltrate and embed in the MMA based resin without any prior wax or decalcification. If interrsted, you could also send me two of these fresh specimens that have not been decalcified or processed into wax and I could cut them for you as well for a proof of concept. I would process, infiltrate and embed one specimen in Acrylosin (MMA resin from Dorn Hart Microedge) and with the other I have another method I try where I could cut the tissue without additional processing or embedding (similar I guess to cryotomy without the freezing process) at a minimum of 10 microns using a laser microtome! Yes, I said a laser microtome! :) What do you say? Contact me back if you are interested and we can discuss the shipping details. Best Regards, Jack Jack L Ratliff Senior Histologist, BioMimetic Therapeutics Chairman, Hard Tissue Committee - National Society for Histotechnology On Aug 19, 2011, at 4:17 AM, Louise Renton wrote: Hi all - a somewhat morbid question for the weekend. A student in the anatomy dept came to me with wax embedded samlpes of demineralized bone from dissection cadavers. The bone is very flinty and difficult to section. Is this perhaps due to the preseravation/embalming process that the bodies undergo? Is there something I can do to alleviate this problem prior to processing? I have tried dewaxing and furher demineralization, but the problem sems to be in the matrix itself-- BTW, using my usual protocol I ahv been able to section elephant tusk - so I think the prob is the bone rather than what I am doing to it best love haev a great weekend Louise Renton Bone Research Unit University of the Witwatersrand Johannesburg South Africa +27 11 717 2298 (tel fax) 073 5574456 (emergencies only) There are nights when the wolves are silent and only the moon howls. George Carlin No trees were killed in the sending of this message. However, many electrons were terribly inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] MMA
Reuel, I would say that you could at least try to use for EM, especially since you already seem to have a polymerized resin block. The biggest issue I would guess would be the overall hardness of your resin block/section so that the section would hold up during the analysis. Also, depending upon the hardness of your block, you may even be able to put your blocks in an oven at 60C for a weekend or so to harden them up even more. It theorized that even though the hardness or softness of a MMA block can be established from the beginning of polymerization (solution concentration of monomer:softner), the block continues to harden over time. I would say subjecting to heat may help to increase this however it may make it more difficult to cut thin sections. In fact, if you let your section roll during microtomy, you may need to adapt to pulling sections flat as the block continues to harden. Hope this makes some sense. Feel free to call me (317-281-1975) if you would like to talk about it over the phone. Jack Date: Thu, 18 Aug 2011 14:06:15 -0500 From: reuel.corne...@tsrh.org To: histonet@lists.utsouthwestern.edu Subject: [Histonet] MMA Can undecalcified bone MMA embedded tissue be use for Electron Microscopy.The tissue was fixed in formalin dehydrated in grades of alcohol, clear in xylene, infiltrated and embedded in MMA (MMA, dibutyl phthalate,perakdox). If not,can anyone have a procedure how to prepare tissue that are embedded in MMA for EM. Is this the same procedure done on a paraffin embedded tissue where you melt the paraffin with xylene then hydrate, wash in distilled water then transfer in osmium ,wash in water, dehydrate, PO,resin. Thank you for your help. Reuel ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] LR White
Teri, You should try using Haupt's Adhesive (www.dornandhart.com) to secure your resin sections. I have never (knocking twice on wood...LOL) lost a section during the staining of thin resin sections when using this product. In fact, I have used this solution for over 14 years and I even subject my methacrylate based resin (Acrylosin SOFT Embedding Solution @ www.dornandhart.com) to deplastification with warmed xylenes prior to staining and still do not lose a section. Also, I have found that wrinkle free is proportional to the softness of the resin block and the section collection, transfer and mounting method. I have attached a few images of specimens embedded with Acrylosin and stained HE, Goldner's Trichrome, and Von Kossa - MacNeal's Tetrachrome for your consideration. Feel free to contact me if you have any additional questions. On a related note, I am giving a teleconference sponsored by the National Society for Histotechnology (NSH) next month (August 17th) as part of their VIR Summer Teleconference Series. During this teleconference I will be talking about the use of resin for undemineralized bone histology. Definitely check this out if you have interest in working with undemineralized bone! Resin Histology: A Practical Approach for Demonstrating Undemineralized Bone Presented by Jack Ratliff, BioMimetic Therapeutics, Inc. As musculoskeletal research progresses with new technological advancements in the areas of biological repair and replacement, histological evaluation continues to play a crucial role in the determination of safety and efficacy for these new treatments. While most will employ traditional and acceptable methods of decalcification and paraffin embedding for the demonstration of these critical components of evaluation, these techniques can sometimes be very challenging and/or impossible when presented with a variety of implant materials or devices. For example, to evaluate safety and efficacy of a metallic device coated with a biological therapeutic at the bone interface, one will need to forego traditional methods of decalcification and seek an undisturbed representation of the specimen by utilizing an embedding media that is both as hard as the specimen and the implant material. Additionally, it may also be important to use a media that will not distort or dissolve the coating. This seminar will address the use of resin histology techniques for the demonstration of undemineralized bone. Topics will include tissue preparation, fixation, processing, infiltration, and embedding/polymerization with acrylic resins. We will also discuss two types of microtomy as related to small and large undemineralized bone specimens and the presence or absence of implant materials. Best Regards, Jack Jack L Ratliff Senior Histologist BioMimetic Therapeutics, Inc. Chairman, Hard Tissue Committee National Society for Histotechnology From: t...@stowers.org To: histonet@lists.utsouthwestern.edu Date: Tue, 26 Jul 2011 10:29:06 -0500 Subject: [Histonet] LR White Is anyone out there using LR White for routine resin embedding, sectioning, and staining? I am interested in learning some tips for mounting sections on to the slide as wrinkle free as possible. Also our HE stains are a little bit pale. Thanks in advance! Teri ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] which conferences?
What type of histology interests you? Research or Clinical? Soft Tissue (paraffin) or Hard Tissue (resin)? I am giving a teleconference sponsored by the National Society for Histotechnolgy (NSH) next month (August 17th) as part of their VIR Summer Teleconference Series. During this teleconference I will be talking about the use of resin for undemineralized bone histology. Definitely check this out if you have interest in working with undemineralized bone! Resin Histology: A Practical Approach for Demonstrating Undemineralized Bone Presented by Jack Ratliff, BioMimetic Therapeutics, Inc. As musculoskeletal research progresses with new technological advancements in the areas of biological repair and replacement, histological evaluation continues to play a crucial role in the determination of safety and efficacy for these new treatments. While most will employ traditional and acceptable methods of decalcification and paraffin embedding for the demonstration of these critical components of evaluation, these techniques can sometimes be very challenging and/or impossible when presented with a variety of implant materials or devices. For example, to evaluate safety and efficacy of a metallic device coated with a biological therapeutic at the bone interface, one will need to forego traditional methods of decalcification and seek an undisturbed representation of the specimen by utilizing an embedding media that is both as hard as the specimen and the implant material. Additionally, it may also be important to use a media that will not distort or dissolve the coating. This seminar will address the use of resin histology techniques for the demonstration of undemineralized bone. Topics will include tissue preparation, fixation, processing, infiltration, and embedding/polymerization with acrylic resins. We will also discuss two types of microtomy as related to small and large undemineralized bone specimens and the presence or absence of implant materials. Jack Date: Tue, 26 Jul 2011 14:17:45 -0700 From: k8...@yahoo.com To: Histonet@lists.utsouthwestern.edu CC: Subject: [Histonet] which conferences? dear all what conference do you recommend in histology and histotechnology feild do you prefer? please provide me with date and a link if avaliable thanx ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Research microtomes
Thanks a lot for your response Adrienne! I think I may be able to help you with this. Is there a time we can talk off-line over the phone? My number is 317-281-1975. Best Regards, Jack On Jul 21, 2011, at 9:49 AM, Adrienne Anderson rennie1...@yahoo.com wrote: This post is actually on behalf of some interns that are working at my company over the summer. Their emails are included on this post, so I'm going to turn it over to them:) But here is their description of what they're doing: In answer to Jack's questions (I thought about replying myself, but didn't want to confuse anyone), we want to make a CEMA array (cutting edge matrix assembly array), which could be done with a variety of tissue types. The step that calls for the special microtome is when you cut the original blocks from the hospital -- meaning variable tissue depth. Though the article mentioned a microtome that could cut 50-2000, we are most interested in the range around 50-150 microns. Thanks, Adrienne From: Jack Ratliff ratliffj...@hotmail.com To: Adrienne Anderson rennie1...@yahoo.com Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu; bush...@rose-hulman.edu bush...@rose-hulman.edu; mlosbo...@gmail.com mlosbo...@gmail.com Sent: Wednesday, July 20, 2011 6:13 PM Subject: Re: [Histonet] Research microtomes Couple more questions. :) What is the tissue and the dimensions of the specimen? On Jul 20, 2011, at 4:57 PM, Adrienne Anderson rennie1...@yahoo.com wrote: Hi Jack, We're trying to cut just plain old FFPE blocks. From: Jack Ratliff ratliffj...@hotmail.com To: Adrienne Anderson rennie1...@yahoo.com Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu; bush...@rose-hulman.edu bush...@rose-hulman.edu; mlosbo...@gmail.com mlosbo...@gmail.com Sent: Wednesday, July 20, 2011 5:02 PM Subject: Re: [Histonet] Research microtomes What type of specimen are you trying to cut? What embedding media are you using? Jack On Jul 20, 2011, at 2:48 PM, Adrienne Anderson rennie1...@yahoo.com wrote: Hello Histo-land, I'm trying to find a microtome that can cut from 50-2000 micron sections. I've only had clinical experience, so I don't know of any such microtome. Any advice would be appreciated! Thanks, Adrienne ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Research microtomes
Couple more questions. :) What is the tissue and the dimensions of the specimen? On Jul 20, 2011, at 4:57 PM, Adrienne Anderson rennie1...@yahoo.com wrote: Hi Jack, We're trying to cut just plain old FFPE blocks. From: Jack Ratliff ratliffj...@hotmail.com To: Adrienne Anderson rennie1...@yahoo.com Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu; bush...@rose-hulman.edu bush...@rose-hulman.edu; mlosbo...@gmail.com mlosbo...@gmail.com Sent: Wednesday, July 20, 2011 5:02 PM Subject: Re: [Histonet] Research microtomes What type of specimen are you trying to cut? What embedding media are you using? Jack On Jul 20, 2011, at 2:48 PM, Adrienne Anderson rennie1...@yahoo.com wrote: Hello Histo-land, I'm trying to find a microtome that can cut from 50-2000 micron sections. I've only had clinical experience, so I don't know of any such microtome. Any advice would be appreciated! Thanks, Adrienne ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Tissue Shriveling in Paraffin
Why not try resin embedding techniques? Less shrinkage than paraffin, more specimen/block stability in cutting, no damaging effects of decalcification and a many times you have a better and more clear morphological representation. I could help you to achieve this if interested, so feel free to contact me as needed (31-281-1975). Jack Jack Ratliff Senior Histologist, Biomimetic Therapeutics, Inc. Chair, Hard Tissue Committee - National Society for Histotechnology On Jun 24, 2011, at 8:27 AM, Michelle Aono aono...@auburn.edu wrote: I was cutting some bone/joint tissue and noticed that the cartilaginous portion was concave/indented, instead of flush with the rest of the block surface. Even as I continued to cut that portion always seemed a little sunken into the block face and all the sections crumbled. I didn't seal the block after I was done and when I came back the next day the entire tissue sample was shriveled and pulling away from the paraffin. I'm new, but in the few bone sections I've done I've never had this happen! Any ideas? ~Shelly ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Sirius Red Stain
Why not save yourself some time and simply purchase the staining kit from Polysciences or Dorn and Hart Micredge. Jack On Jun 23, 2011, at 3:48 PM, Jesus Hernandez jesus.w@gmail.com wrote: Dear all, Does anyone have a protocol on how to prepare Sirius Red stain with known concentration. We are unsure about which concentration to use. We are trying to stain collagen. The Sirius Red is in powder-form. Thank you. Jesse Hernandez University of Texas - San Antonio Department of Biomedical Engineering ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] removing MMA
Bernice, Let me start by first stating that it is easier to melt away the wax from a paraffin block and then go into resin (MMA), than doing it as you have asked. With that said, it has been my experience that this is very difficult to do, but somewhat possible depending upon what you wish to accomplish. The difficulty is dissolving the interior of the specimen that has been completely infiltrated/polymerized and doing so without disrupting the specimen morphology. In fact, dissolving away the exterior takes a lot of time, patience, and solvent. Additionally, you risk changes in morphology, especially on the surface because now everything is loosely supported and the bone will start to become more brittle due to drying with use of the solvent to deplastify. Now to answer your question simply, like dissolves like. If you decide to try this, I recommend to use fresh changes of 100% MMA with agitation to help dissolve away the exterior. As the solution becomes thickened from the deplastification, pour out and start again with fresh 100% MMA. You will need to repeat this process SEVERAL times. Take care not to let your dissolving attempts sit too long (overnight in small amount of solvent) or the solution will start to repolymerize. Basically, the resin will become thick in solution as it is dissolving and settle to the bottom. After a prolonged period of time the thicker layers on the bottom with start to repolymerize slowly. Therefore, before you begin I suggest that you first cut and grind away any excess resin to help you reduce both time and money with the amount of MMA solvent you will waste. A note of caution, acetone will also work but it will damage your specimen over time, so I DO NOT recommend using it. Patience and lots of 100% MMA! Regarding the 50um slides, try Sanderson's Rapid Bone Stain (Dorn and Hart Microedge) and counter with Van Gieson picrofuchsin. Let me know if you have any additional questions and feel free to call me (317-281-1975) if you would like to talk over the phone. Best Regards, Jack PS If this project is for Mahesh, tell him I said hello! :) From: b-freder...@northwestern.edu To: histonet@lists.utsouthwestern.edu Date: Fri, 6 May 2011 13:15:29 -0500 Subject: [Histonet] removing MMA We have a piece of bone in MMA. The researcher wants us to get It out and do paraffin. Is it possible? What will melt the MMA? We also have some 50um slides cut ,that they want to see blood vessels and collagen. Am I going to be able to do an EVG and trichrome or will the plastic inhibit this process? Thanks, Bernice Bernice Frederick HTL (ASCP) Senior Research Tech Pathology Core Facility ECOGPCO-RL Robert. H. Lurie Cancer Center Northwestern University 710 N Fairbanks Court Olson 8-421 Chicago,IL 60611 312-503-3723 mailto:b-freder...@northwestern.edu b-freder...@northwestern.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Von kossa stain
I purchase a kit from Dorn and Hart Microedge and use sodium carbonate-formaldehyde solution to chemically develop. Stain in silver nitrate for 5 min in the dark, 3 fresh DI rinses for 1 min each in the dark, then sodium carbonate-formaldehyde solution for 2 min in the dark, 2 fresh DI rinses (normal light) for 1 min each, 30 seconds in Farmer's Diminisher (Sodium thiosulfate-potassium ferricyanide soluiton to stop reaction) and a running tap water rinse for 10 min. Jack From: dorothy.l.w...@healthpartners.com To: histonet@lists.utsouthwestern.edu Date: Tue, 12 Apr 2011 12:35:18 -0500 Subject: [Histonet] Von kossa stain What is everyone using for their light when developing the silver in the VonKossa stain when you have no sunlight to use? We used to use a 60 watt lamp, but haven't done one for years and am bringing this stain back to our repetiore due to pathologist request. Thanks much! Dorothy Webb, HT (ASCP) Regions Histology Technical Supervisor 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Eosinophilic new bone formation
In decalcified sections of bone, yes the osteoid or dense unmineralized collagen matrix (mostly type I) will stain darker than mature native demineralized bone. Even though the mature bone has been demineralized, it is still more densely compact as compared to the newly formed bone that has not begun mineralization and . In resin embedded undemineralized sections of bone, the contrast is exactly opposite when staining with Von Kossa and counterstaining with MacNeal's tetrachrome and similar to decalcified sections in a Goldner's trichrome where the acid fuchsin stains osteoid darker than the light green does the mineralized bone. So the answer is yes, tissue density is what plays the major role in contrast staining and stain intensity. Jack On Feb 18, 2011, at 6:45 AM, Keller, Pat kell...@ent.wustl.edu wrote: We have always noticed, at least in the middle/inner ear, that newly deposited bone stains darker than mature bone, both with HE and toluidine blue. Is this increased eosinophilic quality due to a lack of mineralization and therefore higher density of osteoid components in the new bone or some other difference in composition of the osteoid? The contrast is quite striking when we observe bone remodeling due to middle ear infections, so I wanted to be able to offer an accurate explanation of why that is... Patricia Keller Sr. Research Tech/Core Histologist Washington University School of Medicine Department of Otolaryngology 4566 Scott Ave Campus Box 8115 St. Louis, Mo 63110 314-747-7166 This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please notify the system manager. This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] stains for visualizing new bone growth
Robin, I typically stain first with Von Kossa and then counter with MacNeal's. This provides a very nice contrast where obviously mature mineralized bone is black and newly formed bone (osteoid) is grayish-green color. Additionally, your marrow space is nicely contrasted with clear visualization of osteoblasts and osteoclasts lining the bone surface. At the microscope this is a one-stop-shop stain for collecting static bone histomorphometry. Another nice contrasting stain is a modified Goldner's trichrome stain. With this stain cell nuclei are stained first with a Weigert's (iron) hematoxylin, then newly formed bone (osteoid) is stained red with an acid fuchsin/ponceau stain, next an orange G cytoplasmic stain covers the rest and a light green SF yellowish stain follows up with a nice green contrast of the mineralized bone. Very clear differentiation between mineralized bone (green) and newly formed bone (red). This stains works very well with auto threshold functions on some histomorph systems as it has a very nice contrast for the software to recognize. Of course Masson's trichrome works as well but it is typically used on decalcified paraffin embedded sections. I have found the Masson's staining kit at Sigma-Aldrich and kits for all the other stains mentioned can be found at Dorn and Hart Microedge. In fact, Dorn and Hart Microedge (www.dornandhart.com) has a lot to offer now with regards to mineralized bone (hard tissue with or without implant materials) and resin embedded histology. Good luck to you and let me know if you have any additional questions. I would also be happy to share images with you if interested. Jack On Feb 1, 2011, at 1:44 PM, Robin Dean robin_d...@compbio.com wrote: Does anyone know of a good stain to use to clearly show new bone growth other than von Kossa stain? Would appreciate any suggestions anyone might have. Thank you, Robin Robin R. Dean, Ph.D. Senior Scientist Study Director Comparative Biosciences, Inc. 786 Lucerne Dr. Sunnyvale, CA (408) 738-8060 robin_d...@compbio.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O
Have you tried Sanderson's Rapid Bone Stain? Try using it first (7 minutes at 60C), then rinse in dH2O (a few dip an dunks at 60C and blot dry), then counterstain with safranin O for 2-5 minutes (room temp - check intensity) and rinse in 100% EtOH (room temp - few dip and dunks and blot dry). If saf o is too intense, you should be able to differentiate a little with 95% EtOH first then do the 100% EtOH step. On a curious note, is there any particular reason why you are cutting thick sections and grinding? You should very easily be able to cut thin sections with a rotary microtome using a tungsten-carbide knife at 5 microns and then you would have greater staining flexibility. Call me if you want to discuss (317-281-1975). Jack On Feb 2, 2011, at 4:18 PM, Herrick, James L. (Jim) herrick.ja...@mayo.edu wrote: Hello everyone, I am trying to stain for cartilage in mouse skull (cross-sectioning with the brain in tact) and wanted to know if anyone might have a good protocol for Safranin O / Fast green or Safranin O / von Kossa? The tissue is embedded in MMA and was cut on a saw microtome and finished down to an approximate 20 to 30 micron tissue thickness - 50 microns including the glue layer (I glue a slide to the block before making the cut and then grind and polish to finish). I tried Weigert's hematoxylin / Safranin O / Fast Green ( I began with Weigert's Hematoxylin, followed by Fast Green and finished with Safranin O), but the Safranin O seemed to overpower the Fast Green (the calcified bone stained more red than green). Any help on this issue would be greatly appreciated as always. Thanks again. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Celestine Blue for Technovit 7200
Why not use Sanderson's Rapid Bone Stain with Van Gieson picrofuchsin? You can get the Sanderson's and one or all of three counterstains for this particular kind of staining in a kit from Dorn and Hart Microedge (www.dornandhart.com). Jack Date: Wed, 20 Oct 2010 16:52:26 -0400 From: sbr...@vetpathservicesinc.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Celestine Blue for Technovit 7200 Hi everyone, I'm searching for a procedure or help w/a stain for thick ground sections using Technovit 7200. The stain in question is a celestine blue/alum hematoxylin w/a Van Gieson counterstain. Anyone have a procedure or recommendations? Thanks in advance, Suzanne sbr...@vetpathservicesinc.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] histomorphometry
At the NSH this year, just this last Sunday in fact, Dr. Tony Villanueva demonstrated this very technique in his workshop. My best advice would be to contact him directly (avillanuev...@cox.net) and see what he can do for you. Another option would be to contact an image analysis vendor like BIOQUANT (nathan...@bioquant.com). Hope this information helps! Regards, Jack On Sep 28, 2010, at 3:25 AM, louise renton louise.ren...@gmail.com wrote: Hi all, I desperately need some advice from an experienced histomorphometristI am trying to translate the old cumbersome visual measurement of using an eypiece grid and doing point counting to an easier computer system - but I am not sure how to do it - any help out there? -- Louise Renton Bone Research Unit University of the Witwatersrand Johannesburg South Africa +27 11 717 2298 (tel fax) 073 5574456 (emergencies only) There are nights when the wolves are silent and only the moon howls. George Carlin No trees were killed in the sending of this message. However, many electrons were terribly inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] protocol for Masson-Goldner trichrome modified by Ulrich
What are your histological endpoints? What is it you wish to demonstrate with this stain? Is it Rodent? Canine? Bone? Cartilage? Microtomed sections? Thick/Ground section? W/ or W/O implants? I can help you with the protocol, but I might be able to provide more assistance. Jack On Aug 20, 2010, at 2:15 PM, Michael Mashore mmash...@vapop.ucsd.edu wrote: Hello everyone, I'm trying to use the Masson-Goldner Trichrome stain, and I came across a paper that used one that was modified by Ulrich. There was no citation on the paper, and I wanted more details to their protocol, i.e. how they made their solutions. The paper is titled Histology and research at the hard tissue-implant interface using Technovit 9100 New embedding technique by Elmar Willbold http://www.sciencedirect.com/science?_ob=ArticleURL_udi=B7GHW-50CVR3J-1_u ser=4429_coverDate=06%2F25%2F2010_rdoc=1_fmt=high_orig=search_sort=d_d ocanchor=view=c_searchStrId=1436969926_rerunOrigin=google_acct=C5960 2_version=1_urlVersion=0_userid=4429md5=ac8acdf1a211881097275a05690a4259 #cor1 and Frank Witt. Does anyone know more details on this stain? We are using it on hard tissue embedding, where we used methylmethacrylate. Thanks for the help! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Resin embedded tissue
Valerie, I use just regular glass slides, coat them with Haupt's adhesive, roll the sections flat and then press and dry the sections to the coated slide using a slide press and oven at 50C. In 13 years of hard tissue resin histology, I have never (knocking twice on wood) lost a section or had a section lift up in any area during staining using this method. You will get a degree of background with certain stains (hematoxylin, analine blue, etc) because it is a gelatin based adhesive, but these problems are easily resolved with a little overstaining and acid alcohol rinse. If interested you can get the Haupt's and slide press from Dorn and Hart Microedge (www.dornandhart.com). I have also been told that they have a couple of new kits coming out by the end of the summer - a resin embedding kit (using Perkadox as a catalyst) and thin section microtomy kit (one for a rotary microtome and one for a sledge or Polycut microtome) that includes everything needed to section resin blocks. Look them up and contact Bill Hart for more information. Jack On Jul 15, 2010, at 3:09 AM, Tilston, Valerie v.tils...@liverpool.ac.uk wrote: Hi, Does anyone have experience of cutting resin embedded bone and if so what type of slides do people use? We are having problems with sections falling off or the bone not remaining flat on the slide! Many thanks in advance, Val ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] MMA Formulation for EXAKT Grinding
Nikki, The MMA formulation you describe is one that yields a very soft block suitable for thin section (4-6 microns) microtomy. I have heard where people use this formulation for undemineralized bone and maybe even for stent work, but the softness of this formulation concerns me for the stents given the reduced stability. For me personally it is not what I use for both of these specimen types, but that is for another discussion. It is important to note that the volume of catalyst is proportional to the total volume of solution and thus this ratio (expressed as w/w) is directly proportional to the reaction product. Now to make things a little more confusing, the reaction product is influenced by air temperature, the size/density of the specimen, the total volume of reaction product, and sometimes the embedding container and void space above the solution level and container lid. Furthermore, since this reaction or polymerization of resin is an exothermic reaction, the rate (expressed as a unit of time) at which the reaction reaches the actual point at which polymerization initiates (v-max) also then influences the amount of heat that is generated from the reaction. This then is proportional to the quality of polymerization that can be seen as either a hard clear desirable block or and over polymerized, bubbled mess!!! It is my opinion that the bubbles in your specimen blocks are related to the build up of pressure in your container and caused by a rapid polymerization of your specimens by the use of the heated water bath (as per you concentration of catalyst to MMA/DBP solution) and lack of void space to buffer or diffuse excess heat. My feeling is that you are using too much catalyst in conjunction with the heat of the water bath to polymerize these specimens. Also, what is the volume of the solution you are polymerizing, how close are your specimen molds to each other in the water bath, and is the water level of the water bath at or above the embedding solution level in the specimen container? The heat generated from one specimen can sometimes add to the heat generated by another in close proximity. This then results sometimes in an over polymerization of one specimen (too much heat generated in the reaction) and no polymerization of another (absence of heat to drive the reaction). Here are my suggestions: 1) If you need specimens polymerized immediately the next day, take care to space out your specimens further apart in the water bath. Also, try turning down the water bath to reduce the secondary heat used to drive the reaction. If none of this works, then look at reducing the amount of catalyst used (may want to do this first and keep everything else the same). 2) If you can spare a few days, don't change a thing with the embedding solution, try switching your molds to polypropylene containers and leave them out on the counter at room temperature (22-23C) for 2-3 days until they polymerize. Hope this helps and it wasn't too confusing. Jack On Jun 25, 2010, at 3:46 PM, Wahlberg, Nikki nikki.wahlb...@bsci.com wrote: Hello Everyone, I was wondering if you could help me with my MMA formulation. I have been using a formulation that I found in a published paper. My current embedding formulation is 80ml MMA, 20ml Dibutyl Phthalate and 3g Benzoyl Peroxide. The samples are embedded after three days of infiltration, one change per day, with the formulation of 80ml MMA and 20ml Dibutyl Phthalate. Lately have noticed that there is a pressure build up in the vials. I have had a few vials burst almost immediately once placed in the heated waterbath. I am filling the glass vials full with the embedding solution, capping them and then placing them in a water bath in a 37 degree oven. They are completely polymerized by the next morning. I am also getting bubbles in the plastic when polymerized. I have two questions: Is there any way to get rid of the bubbles in the plastic and of more concern what do you think is causing the pressure build up? I would really appreciate any help that you can provide. Thank you, Nikki ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] how to prevent foldings on femoral head cartilage tissue
Reuel, Do the opposite and turn up the temp on the waterbath and let the section float out a little before the wax starts to break up. You can even gently use the forcepts to tease out any folds and this will definitely help to release any wrinkles in the cartilage. Then, make sure the section is free of large droplets of water before transfer of the slide to the slide warmer. Next, let slide sit on slide warmer until the wax is melted and then bake the section for a few minutes at a higher temperature to firmly secure the section to the glass slide. Bob Skinner at UAMS showed me this technique at the beginning of the year and it works nicely, especially for even larger human femoral heads! Jack Date: Thu, 17 Jun 2010 12:10:10 -0500 From: reuel.corne...@tsrh.org To: histonet@lists.utsouthwestern.edu Subject: [Histonet] how to prevent foldings on femoral head cartilage tissue Hello histonetters especailly hard tissue group I have a pig femoral head bone tissue embedded in paraffin and I have a hard time getting rid of the folding problem. I tried to remedy by lowering my temperature to 38 C and putting them in 5% alcohol before placing them in water bath I still have a lots of folding formation on some areas of the cartilage. Is there any other technique to remedy this problem. I appreciate your help. Thank you. reuel ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Hotmail has tools for the New Busy. Search, chat and e-mail from your inbox. http://www.windowslive.com/campaign/thenewbusy?ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_1___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] intervertebral disc
Jennifer, What do you wish to accomplish histologically? Do you only wish to see the disc material? Do you care about the cranial and caudal vertebral bodies? Are you wanting to perform IHC? Please tell me a little more so that I can provide you with a more detailed options. I am assuming that this is an IVD project and maybe you only wish to see the treatment of the nucleus pulposus? Jack From: jander...@halozyme.com To: histonet@lists.utsouthwestern.edu Date: Wed, 16 Jun 2010 13:30:51 -0700 Subject: [Histonet] intervertebral disc Greetings. What is the best way to process intervertebral disc tissue from pig? Right now I have 4 intact vertebrae with attached discs in formalin. Should I just use a sharp knife? Thanks so much for your expertise. Jennifer M. Anderson, Scientist Halozyme Therapeutics, Inc. 11388 Sorrento Valley Road San Diego, CA 92121 858-704-8333 (office) jander...@halozyme.commailto:jander...@halozyme.com The information transmitted in this email is confidential and is intended only for the person(s) or entity to which it is addressed. Delivery of this message to any person other than the intended recipient(s) is not intended in any way to waive confidentiality or any applicable privilege. Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by individuals or entities other than the intended recipient is prohibited by Halozyme and may be in violation of applicable laws. If you received this in error, please contact the sender and delete/destroy this email. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Hotmail has tools for the New Busy. Search, chat and e-mail from your inbox. http://www.windowslive.com/campaign/thenewbusy?ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_1___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] NSH Hard Tissue Forum - August 14th, 2010 in Philadelphia, PA
The Hard Tissue Committee of the National Society for Histotechnology is proud to present a one day Hard Tissue Forum. This first of its kind event will be held Saturday, August 14th, 2010 in Philadelphia, PA, from 8:00 am to 5:00 pm at the Doubletree Hotel Philadelphia. Join us as we seek to further our knowledge and understanding of the histology and analysis of bone and how this information can better serve in the diagnosis of bone related diseases and the efficacy and safety of therapeutic treatments. Topics include: Bone Biology 101; In Vivo Models for Musculoskeletal Research; Concepts for Specimen Management of Samples for Decalcified Paraffin Processing; Resin Histology – A Practical Approach for Demonstrating Undemineralized Bone; Skeletal Analysis with MicroCT – What You Need to Know and Why You Need To Know It; and Bone Histomorphometry. Don’t miss out on this one time compilation of information solely dedicated to bone as presented from both scientists and histotechnologists actively working in the field. This forum will also be a perfect opportunity to network with professionals within the “hard tissue niche”. We hope to see you in August! Click Here for More Information and To Download a Copy of The Registration Brochure or contact Jack L Ratliff, NSH Hard Tissue Committee Chair @ 317-281-1975 or jratl...@biomimetics.com. Best Regards, Jack Ratliff NSH-Hard Tissue Committe Chair _ Hotmail is redefining busy with tools for the New Busy. Get more from your inbox. http://www.windowslive.com/campaign/thenewbusy?ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_2___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Mounting Medium Issues
Jim, I must say that I have used Eukitt exclusively for over 13 years and the only problem I have had with Eukitt is when I do not use enough and especially when coverslipping thicker sections. It is xylenes based so maybe you are experiencing excessive evaporation during drying??? If you are diluting the Eukitt with xylenes so that it flows better (not as viscious) and reduces the chance for air bubbles, this might cause excessive shrinkage and/or the problems you are experiencing. Jack On May 14, 2010, at 3:22 PM, Herrick, James L. (Jim) herrick.ja...@mayo.edu wrote: Hello everyone, Hope you have all had a good week!! Has anyone had any negative experiences using Eukitt as your mounting media? We embed our specimens (human - iliac crest, animal - femurs, tibias, vertebrae, etc.) in GMA and MMA and have noticed over a period of time that a fairly large number of our slides are beginning to show signs of the medium breaking up below the cover slip. It looks as if there are large air pockets left behind. We are not yet sure why this is happening. Would any of you geniuses have a resolution for us or know of a better mounting media to use with plastic embedded specimens - brightfield and fluorescence? Thanks for your expertise. It is very much appreciated. Have a great weekend!! Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] large fibrous bone tissue
Louise has very good advice here as related to paraffin processing of this tissue. I may even add to soak the block a little more before taking the final sections. However, have you ever thought of processing into MMA resin? If you have these capabilities you may be very pleased with the results and find the microtomy less problematic. Let me know if or how I can be of assistance! Jack On May 15, 2010, at 4:30 AM, louise renton louise.ren...@gmail.com wrote: I have found this helps. 1. Embed the tissue in a dep mould, as this provides more stability, then 2. Face the block 3.. leave in -20 deg freezer overnight 4. remove from freezer and cut sections 5. If you have multiple blocks to work with, leave them in the freezer until ready to cut regards On Fri, May 14, 2010 at 7:42 PM, Reuel Cornelia reuel.corne...@tsrh.org wrote: How do you process a fibrous bone tissue ( 7 mm thick). We have use Paraffin Type 9 from Richard allan Scientific to embed works well with our bone femur( 7 mm) when cutting but on fibrous bone it does not give us a good result in cutting the blocks. It is like cutting a uterus tissue but a little bit harder. Please give me your opinion on how to remedy this kind of tissue not mentioning double embedding method or plastic. Thank you. Reuel Cornelia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Louise Renton Bone Research Unit University of the Witwatersrand Johannesburg South Africa +27 11 717 2298 (tel fax) 073 5574456 (emergencies only) There are nights when the wolves are silent and only the moon howls. George Carlin No trees were killed in the sending of this message. However, many electrons were terribly inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] need tips for cross-sectioning of cortical bone
Why not embed in resin (MMA) and take thicker sections and then grind/ polish them down? If you went this route, you could then use flourescent labels and quantify mineral apposition rate and bone formation rate. Let me know if you are interested. I can help you get started and direct you to low cost equipment options. Jack On Apr 22, 2010, at 9:58 AM, Connolly, Brett M brett_conno...@merck.com wrote: A colleague is having trouble getting wrinkle-free sections of decalcified, paraffin embedded femur. Any tips?? Thanks, Brett M. Connolly, Ph.D. Molecular Imaging Team Leader Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html ) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Immuno staining on plastic sections
A few years ago I took a workshop at the NSH presented by Neil Hand. He basically provided an account of his experience in working with something like over 100 antibodies used on resin (MMA) embedded bone and possibly other tissues. Maybe you can look his information up on the NSH website and contact him directly? With regards to an etching step, there are several posts that describe everything from HCl to formic acid to sodium ethoxide. In fact, I just found this posting from Gayle Callis back in 2004. Hope this helps! Jack [Histonet] Repy on immunostaining with Technovit 8100 / glycol methacrylate Gayle Callis gcallis @t montana.edu Thu Aug 12 10:13:55 CDT 2004 GMA is not a very ideal embedding media for immunohistochemistry, you would be far better off with paraffin using this antibody. GMA cannot be removed once polymerized nor sodium ethoxide (generally reserved for electron microscopy resins) etched with much success, you would be better off embedding in methylmethacrylate and remove the plastic entirely. This has been done with great success by Neil Hand (he has publications) using warm xylene, but he used stringent pressure cooker retrieval and worked with human tissue. The problem with GMA is it prevents the immunoglobulins from reaching antigenic sites, as the GMA is hydrophobic plus it can't be removed once polymerized. It will soften in the presence of water. Also, as GMA polymerizes it becomes very hot due to exothermic reaction unless you control this temperature by letting your blocks polymerize on ice in a refrigerator?? GMA with IHC problems have been discussed at length on Histonet many times, go to Histonet archives and search at www.histosearch.org. Personally, I don't think the product suggestion is correct, as it only suggests but does not say it WILL work. That is probably the reason you don't find it in the literature! Many people have experienced failure of IHC on GMA embedded tissues. I think some have had success with immunofluroescence using immunoglobulins and not a lot of antibodies either. It was a tedious stringent protocol described in a symposium talk. I think you will find in Histonet commentary, that most people attempting IHC/GMA suggest going to another embedding media. Also, CD68 ED1 is a Mouse antiRat, rather than a rat antiMouse (clone FA-11). ED1 and other rat macrophage markers, ED2, ED3, have been discussed on Histonet as FFPE tissues including retrieval for IHC. The staining was very straightforward as was retrieval described and solvents used plus heat of paraffin processing did not damage antigen. ED1, when used in on rat tissue, works well on paraffin sections, although we prefer frozen sections to avoid all aldehyde fixation and no retrieval. When we do frozen sections, the ED1 is diluted out 1:3000 or so, it will not be so dilute for paraffin sections and we detected with secondary to mouse IgG1 isotype (Southern Biotechnology) SA-HRP, and AEC chromogen. Staining pattern is spectacular in a rat spleen, normal positive control. Gayle Callis MT,HT,HTL(ASCP) Research Histopathology Supervisor Veterinary Molecular Biology Montana State University - Bozeman PO Box 173610 Bozeman MT 59717-3610 To: histonet@lists.utsouthwestern.edu Date: Fri, 9 Apr 2010 09:11:59 -0400 From: brusk...@aol.com Subject: [Histonet] Immuno staining on plastic sections We are trying to do immuno staining on plastic sections and are not having much luck. Does anyone have any experience with this? We are embedding in technovit 8100. It is a device with encapsulated VEGFR cells. I have been told that we might have to do an etching step. Any help would be greatly appreciated. Bruce ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Sanderson's Bone Stain
This stain is now available via Dorn and Hart Microedge - www.dornandhart.com Jack From: bustaman...@uthscsa.edu To: histonet@lists.utsouthwestern.edu Date: Tue, 2 Mar 2010 10:01:19 -0600 Subject: [Histonet] Sanderson's Bone Stain Hello anyone out there, A couple of years ago, I ordered Sanderson's Rapid Bone Stain from Surgipath. Since they have gotten bought out, they no longer carry this stain. Does anyone know where I can order it? Thanks ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Alternatives to BioQuant for Bone Histomorphometry
Adam, What stain are you using for your quantitation and are you trying to perform measurements via the thresholding feature? Jack On Feb 5, 2010, at 4:38 PM, Adam . anonwu...@gmail.com wrote: Hi all, I am looking for an alternative program to BioQuant for bone histomorphometry. We need to quantify the number of osteoblasts / osteoclasts per bone surface area as well as the percent surface area occupied by those cells. We have a computer with BioQuant on it available, but we find the software to be incredibly clunky and often nearly impossible to use. Based on my limited attempts to use it, it very well might rank as one of the worst user interfaces I've ever seen, and I was trained in computer science and have seen my fair share of horrible software (I'm looking at you, Lotus Notes). Anyhow, any suggestions on a (preferably cheap / free) replacement for doing simple analysis or how to make BioQuant less painful would be very helpful. Thanks, Adam ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] sharpen permanent knife blades
I will also add that DDK and Dorn and Hart Microedge also sharpen knives. One should also check the pricing as they all vary. Jack Sent from my iPhone On Dec 22, 2009, at 8:07 AM, Smith, Allen asm...@mail.barry.edu wrote: C.L. Sturkey (www.sturkey.com) in Lebanon, Pennsylvania, sharpens permanent microtome knives. They also have two grades of disposable blades. Allen A. Smith, Ph.D. Professor of Anatomy Barry University School of Podiatric Medicine -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet- boun...@lists.utsouthwestern.edu] On Behalf Of MARCELLYN STONE Sent: Tuesday, December 22, 2009 9:00 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] sharpen permanent knife blades Please help:-). I am looking for information on anyone who sharpens permanent knife blades. Thanks Marcy CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may contain confidential and privileged information for use by the designated recipients named above. They are intended solely for these recipients. If you are not the intended recipient, you are hereby notified that you have received this communication in error and that any review, disclosure, dissemination, distribution or copying of it or its contents is prohibited. If you have received this communication in error, please notify Calvert Memorial Hospital immediately by telephone at (410) 535-8282 and destroy all copies of this communication and any attachments. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Trichrome stain on MMA sections
You can do HE and VVG quite nicely on this type of MMA embedded tissue. Maybe you could also try a Sanderson's (methylene blue) with the Van Gieson (acid fuchsin w/ picric acid) counterstain??? On Dec 21, 2009, at 11:51 PM, Randall Carpenter rjc...@usiwireless.com wrote: Dear Histonet, I was wondering what the best Trichrome stain might be for sawn and ground sections of large stent/artery in methylmethacrylate. I am also looking to stain elastic fibers. Any suggestions? Thanks. Randy Carpenter ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] sharpen permanent knife blades
I agree with most of what you have stated and espcially with sticking with one company once you get things moving in the right direction. However, if one truly knows how to properly set and/or adjust cutting angles at the microtome, I would definely keep cost at a high consideration if quality is uniform across vendors. Of course, everyone also has various demands as related to workload volume and study turnaround time. These variables should also be considered and again, especially if product quality is not compromised. Jack Ratliff Date: Tue, 22 Dec 2009 15:36:43 + From: mucra...@comcast.net To: ratliffj...@hotmail.com CC: Histonet@lists.utsouthwestern.edu; mst...@cmhlink.org; asm...@mail.barry.edu Subject: Re: [Histonet] sharpen permanent knife blades Different companies have varied angles on how a knife is sharpened for their equipment. A knife sharpened by one company will generally be somewhat different in the angle of the edge than another when you begin cutting. Some may have a flatter edge or a fatter edge at the top cutting portion. These are not always easy to see and can cause issues with sectioning although sharpness is the same or very close. Using several different companies can lead to problems in adjusting the knife angle for sectioning from one to another. I would select one company and stay with it to avoid these issues. I found a company I preferred and then stuck with them rather than fight the problems over a few dollars. Pam Marcum - Original Message - From: Jack Ratliff ratliffj...@hotmail.com To: Allen Smith asm...@mail.barry.edu Cc: Histonet@lists.utsouthwestern.edu, mst...@cmhlink.org Sent: Tuesday, December 22, 2009 9:06:03 AM GMT -06:00 US/Canada Central Subject: Re: [Histonet] sharpen permanent knife blades I will also add that DDK and Dorn and Hart Microedge also sharpen knives. One should also check the pricing as they all vary. Jack Sent from my iPhone On Dec 22, 2009, at 8:07 AM, Smith, Allen asm...@mail.barry.edu wrote: C.L. Sturkey (www.sturkey.com) in Lebanon, Pennsylvania, sharpens permanent microtome knives. They also have two grades of disposable blades. Allen A. Smith, Ph.D. Professor of Anatomy Barry University School of Podiatric Medicine -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet- boun...@lists.utsouthwestern.edu] On Behalf Of MARCELLYN STONE Sent: Tuesday, December 22, 2009 9:00 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] sharpen permanent knife blades Please help:-). I am looking for information on anyone who sharpens permanent knife blades. Thanks Marcy CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may contain confidential and privileged information for use by the designated recipients named above. They are intended solely for these recipients. If you are not the intended recipient, you are hereby notified that you have received this communication in error and that any review, disclosure, dissemination, distribution or copying of it or its contents is prohibited. If you have received this communication in error, please notify Calvert Memorial Hospital immediately by telephone at (410) 535-8282 and destroy all copies of this communication and any attachments. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Sanderson's rapid bone stain + Acid fuchsin
Regarding the counterstain for use with Sanderson's Rapid Bone Stain, I use the Van Gieson picrofuchsin in that it will stain older, mature, mineralized bone red. As an additional consequence of the VG counterstain, the originally blue stained dense collagen (new bone/ osteoid) from the SRBS will change to a greenish color from the reaction of the yellow picric acid (present in the counterstain). If you think about it, it makes perfect sense due to the basic reaction of combining blue and yellow to yield green. Additionally, the intensity of the red from the acid fuchsin content of the counterstain is directly proportional to the mineral density of the bone due to the acid in the solution acting to lightly etch the mineralized bone. Also, think of the specimen in 3D, you could expect shades of red depending on the plane of sectioning within the specimen and depending upon mineral deposition at the surface of trabeculae. Therefore, I believe that the dye-tissue interaction you are inquiring about is associated to mineralization or calcium concentration. Hope this helps to answer your question. Jack On Dec 21, 2009, at 1:25 PM, sk...@illinois.edu wrote: Hi All - I am trying to figure out if the combination of Sanderson's rapid bone stain and acid fuchsin as the counterstain (or methylene blue + basic fuchsin) results in pink/red staining of biologically formed apatite (apatite not associated with collagen produced by osteoblasts)? An associated question is if the dye-tissue interaction between acid or basic fuchsin and mineralized bone is due to dye-collagen interactions? If anyone has any experiences or references that may help, please let me know. Thank you for your help, Sheeny ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Bone labelling for Microdamage
Karie, If you are looking for microdamage or are using basic fuchsin, I have been taught to bulk stain with 1% basic fuchsin in your processing solutions (i.e. 1% BF in 70% EtOH, 1% BF in 80% EtOH, 1% BF in 95% EtOH, and 1% BF in 100% EtOH) and then finish as you would with the remaining steps for your MMA protocol (i.e. xylenes, MMA +DBP, etc.) to achieve a fully polymerized bulk stained specimen. You then cut, grind, and polish as required for use with the EXAKT system. I was told the reason you process with the BF is because the cutting and grinding causes additional microdamage and if you stain first the additional damage will not be stained. If you are talking about labeling bone for calculations of BFR or MAR and the animal did not receive timed injections of calcein, alizarin, tetracycline, or some other fluorescent bone label before necropsy, then you could try doing a 0.6% FA etch for 30 seconds followed with Sanderson's Rapid Bone Stain for 3 minutes, brief tap water rinse, and blot dry. What is the exact specimen you are working with and what do you specifically wish to demonstrate Jack On Dec 1, 2009, at 10:04 AM, Karie Reaser krea...@vet.upenn.edu wrote: Is it ok to process cortical bone, embed in MMA, section and grind on the Exakt system. Last do the bone labelling stain? Or do I have to do the bone labelling stain prior to embedding, sectioning and mounting on slides? Any suggestions would be greatly appreciated. -- Karie L Reaser A.S. New Bolton Center University of Pennsylvania School of Veterinary Medicine Comparative Orthopedic Research Laboratory 382 W Street Road Kennett Square, PA. 19348 Phone:610-925-6278 Fax:610-925-8120 Email:krea...@vet.upenn.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] MMA Sections
Karen, How about coating your slides with Haupt's. I have used this solution exclusively for all my undemineralized thin section bone work. Also, I only need to dry my slides overnight using a dense aluminum slide press (available via Dorn and Hart Microedge). Basically, the slide press acts to evenly flatten and press the sections to the Haupt's coated slide and also evenly heat all the slides together. Because of it's heat conducting properties, the press acts as a mini-oven within the oven to speed up the drying and section adhesion process. Please feel free to contact me if you have any questions regarding this or any other resin related topic. Regards, Jack Ratliff NSH Hard Tissue Committee Chair On Dec 1, 2009, at 9:52 AM, Karen Hughes hugh...@upstate.edu wrote: I am currently embeding 2cm sections of adult rat spine in MMA including muscle, bone and spinal cord. I then cut 5um sections using a tungsten carbide D profile knife on a manual microtome and mount the sections on APES coated slides, press them in a vise between tongue depressors, and bake them @ 41'C for a min. of 48hrs. My problem is that when I de-placticize the slides in Xylene (4 changes, 2x5min, 2x10min) the spinal cord falls off the slide, while the bone stays secure. I have also tried PolyLysine coated slides, SuperFrost Plus, and Probe On Slides. All with the same results. Does anyone have any experience with this? Any and all Suggestions would be greatly appreciated. Karen Karen S. Hughes Research Support Specialist SUNY Upstate Medical University Phone (315)464-8585 hugh...@upstate.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] course recommendation
Take a look at the National Society for Histotechnology (www.nsh.org). There are several State and Regional Society meetings coming up in the spring of 2010 and then of course there is the National Symposium/Convention in the Fall of 2010 in Seattle, WA. Jack Ratliff NSH -Hard Tissue Committee Chair Date: Fri, 20 Nov 2009 12:57:36 -0500 From: mti...@trudeauinstitute.org To: histonet@lists.utsouthwestern.edu Subject: [Histonet] course recommendation I am looking for a conference/course to attend in 2010. I am experienced in IF and IHC and would like to learn more about any new techniques and products that might be available. Does anyone have a recommendation for a course or a conference in the US? I am not a certified HT but I pretend to be one:) Thanks for any suggestions!! Mike ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Sanderson's RBS
Andrew, I will be updating the Hard Tissue Committee members regarding this very soon but this is very funny that the subject has come up yet again. In fact, I have received a few phone calls over the past couple of weeks on the subject and ironically two yesterday. Addittionally, this was of great concern to me because I too depend upon this product and of concern to several HTC committee members when we met at NSH and discovered Surgipath was no longer going to carry the stain. Nevertheless, I have been told that this stain will now be available via Dorn and Hart Microedge Evidently Dorn and Hart is in the process of expanding beyond the services of providing and resharpening knives. In fact, I spoke with the son (Bill Hart) and I was told that Sanderson's Rapid Bone Stain is just the start of many exciting things they will begin to offer hard tissue folks in the coming weeks and months. Rest assurred that I will keep committee members informed of the availability of this stain and this new direction of DH as more information becomes available. Jack Ratliff NSH Hard Tissue Committe Chair On Nov 12, 2009, at 4:28 AM, Prior, Andrew andrew.pr...@smith-nephew.com wrote: I'm looking for a new supplier for Sanderson's Rapid Bone Stain as Surgipath have stopped selling it (at least in Europe). Does anyone know of an alternative supplier or an equivalent stain I could substitute in? [Currently use the RBS on 15µm ground Technovit 7200VLC resin sectio ns, with acid fuchsin counter-stain.] Thanks in advance Andrew Andrew Prior Histologist Smith Nephew Research Centre Confidentiality. This electronic transmission is strictly confidential to Smith Nephew and intended solely for the addressee. It may contain information which is covered by legal, professional or other privilege. If you are not the intended addressee, or someone authorised by the intended addressee to receive transmissions on behalf of the addressee, you must not retain, disclose in any form, copy or take any action in reliance on this transmission. If you have received this transmission in error, please notify the sender as soon as possible and destroy this message. Smith Nephew UK Registered in England and Wales No.4421171 with registered office at 15 Adam Street, London WC2N 6LA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] osteoblast count
I am not sure about after staining with IHC, but if you stain with a toluidine blue or MacNeal's tetrachrome you can easily identify osteoblasts. Jack Date: Fri, 16 Oct 2009 07:56:40 -0700 From: dr.hatemsa...@yahoo.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] osteoblast count HI I have a problem with counting osteoblast in IHC staining slides is there a way to differentiate osteoblast from other bone cell types. thank you very much Hatem ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bone sections
Are you working with resin or paraffin sections? Jack Sent from my iPhone On Aug 18, 2009, at 6:50 AM, Williams, Sean s.a.willi...@liverpool.ac.uk wrote: Hi Everyone Could any one tell me how they keep bone sections adhered to slides when staining. I'Ve tried polysine slides, pva glue slides, different ways of drying the sections and for different lenghts of time but nothing seems to work for every section. I'm only staining them with HE. Thanks Marie O'Brien (liverpool vet path - histology) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] cement in bone specimen
Peggy, If the cement you mention is or similar to PMMA, then you will not be able to cut thin sections using the sliding/sledge microtome. If I was you, I would not decal it at all as it will only make the bone less dense than the cement and will definitely waste your time if you embed it in paraffin to try and cut later. Your best and only chance at success here is to embed undemineralized in resin and cut thick or ground sections that can be polished to a desired thickness. If you would like to discuss this further, please give me a call at 317-281-1975. Best, Jack Ratliff Date: Thu, 6 Aug 2009 15:43:16 -0400 From: mdica...@kaleidahealth.org To: histo...@pathology.swmed.edu CC: Subject: [Histonet] cement in bone specimen Histonetters, We sliced a humerus bone on a band saw which was filled with cement from a previous surgery and my boss would like me to try to cut it on a sliding/sledge microtome. (The knife remains stationary) Is this possible to do? Presently, it is fixing and then I will decal it but I'm wondering if I'll be wasting my time and effort. I would appreciate anyone's experience with this. Thank you. Peggy DiCarlo HT (ASCP) Orthopedic Bone Lab Buffalo General Hospital Buffalo, NY 14203 716-859-1293 2009 Best Places to Work Winner Visit our careers page at www.kaleidahealth.org/careers CONFIDENTIALITY NOTICE: This email transmission and any documents, files, or previous e-mail messages attached to it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, or a person responsible for delivering it to the intended recipient, you are hereby notified that any further review, disclosure, copying, dissemination, distribution, or use of any of the information contained in or attached to this e-mail transmission is strictly prohibited. If you have received this message in error, please notify the sender immediately by e-mail, discard any paper copies, and delete all electronic files of the message. If you are unable to contact the sender or you are not sure as to whether you are the intended recipient, please e-mail ist...@kaleidahealth.org or call (716) 859-. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] VonKossa's calcium stain
What is your tissue of interest? Why not do the Von Kossa stain first and then counterstain with MacNeal's tetrachrome. This way you employ the use of a metachromatic stain for the rest of the tissue instead of just a nuclear staining hematoxylin. Jack On Jul 16, 2009, at 9:34 PM, karine cadoret kcado...@amc.edu.au wrote: Hi, When doing a VonKossa stain in order to demonstrate calcium in tissue, does it matter much if I use Mayer's hematoxylin instead of Ehrlich's hematoxylin (which takes 6 months to ripen) ? Also, can I simply use homemade scott's tapwater for blueing instead of using a lithium carbonate solution ? Thank you for your help, Karine Cadoret Fish health laboratory manager National Center for Marine Conservation and Resource Sustainability Newnham, TAS Australia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] osteoids
My recommendation to you is to focus on your undemineralized bone sections embedded in MMA. These sections will provide you with the most information. If you do a Goldners stain you can very accurately measure BV/TV and clearly quantitate osteoid volume. Bone will stain green from the SF yellowish staining and osteoid red with the acid fuchsin/ponceau. I feel the only limitation with this stain is clearly assessing your bone cells. If you stain with a Von Kossa/MacNeal's tetrachrome you can achieve the above (bone = black, osteoid = greyish-green) and you can also clearly assess osteoblast (blue) activity. This stain also helps to identify osteoclast presence but hard to accurately determine activity without a TRAP stain follow up. Write me back with any additional questions you may have and when I get to the lab I will forward you a couple of protocols and additional information! Best, Jack Sent from my iPhone On Jul 2, 2009, at 7:59 AM, Manav Mehta manav.me...@charite.de wrote: Dear Histonet'ers, I am trying to stain for Osteoids in a fracture callus of rat femurs. I have MMA and Paraffin sections (4-5 um) / blocks already made. However, I am not sure what stain I should use. Peers have suggested that a Movat pentachrome should work, while some have suggested Goldner, and silver staining. I am not sure how to proceed at the moment. I would appreciate any suggestions with possibly a protocol on the staining for osteoids. It would also help if you provided references on this stain or details on the stain. Life experiences while working with these stains are also welcome. The Goal is to characterize the osteoids in the callus (surface, thickness, osteoblast numbers). Possibly relate them to osteoblastic and -clastic activity. Thanks, Manav Mehta ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Bone saw
IMEB also has a Bone Band Saw. http://www.imebinc.com/Item/BBS-82203.htm Jack Date: Thu, 21 May 2009 09:05:50 -0700 From: cb...@memorialcare.org To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Bone saw Good Morning, I was wondering if anyone can help me find a really good saw for bones (femoral/humeral heads mainly). We currently have a MarMed bone saw that works great for knees and such but it's just not strong enough for the femurs. Thank you, Christine Bark HT(ASCP) CM Lead Histotech, Pathology Saddleback Memorial Medical Center 949-452-3548 cb...@memorialcare.org __ Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] bone tissue
Haupts Gelatin works really well at a 1:1 ratio (Concentrate:50% EtOH). The only limitation is background staining from say a hematoxylin counterstain after the IHC. You can purchase the concentrate from Fisher (Haupts Fixative #785-71). Jack Date: Thu, 21 May 2009 10:12:23 -0500 From: reuel.corne...@tsrh.org To: histonet@lists.utsouthwestern.edu Subject: [Histonet] bone tissue Can somebody help me with my bone tissue to remain intact during antigen retrieval for IHC staining. I have used subbed slides but It did not help me. Is there more better adhesive. Does Haupts Gelatin works better? I know this is a hundred year old question and hopefully this problem have been resolved since my undated knowledge could remember. I would like to thank everybody for responding to my last e-mail on gross photography. It gives me more idea what to purchase. Thanks again histonetters and I really appreciate your help. Reuel Cornelia, BS MT, AMT Cellular Pathology Texas Scottish Rite Hospital for Children Welborn Street Dallas, TX 75219 Tel: 214-559-7766 fax: 214-559-7768 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Bone!
I do not routinely perform decalcified bone preparations as I predominantly utilize undemineralized resin (MMA) applications, so maybe someone else can better respond to this question. However, I would say that you could store your bone in 70% EtOH until you are ready to process, but the length of time in solution storage may cause you problems down the road since you have already decalcified. I think that a prolonged storage time might make your bone hard againif that makes any senseand cause you a fit later when you go to the microtome. Typically, I store undemineralized bones at either 10% NBF or 70% EtOH depending upon what I care to see at the microscope. Jack Date: Thu, 21 May 2009 10:46:59 -0300 From: redw...@ucb.br To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Bone! Friends, Can someone recommend to me how to store bones (juvenile rat femur) that I have fixed and decalcified, but am not yet ready to cut (they're for vibratome, so not embedded). Thanks! Redward. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Affordable microtome knife reconditioning in the EC?
I don't know about Europe, but here in the US there are a few vendors that provide these services (Dorn and Hart Microedge, Sturkey, and Delaware Diamond Knives). I am not sure of the pricing for all three of these vendors, but for the past 11 years I have sent my D-profile rotary wedge and sledge/polycut tungsten-carbide knives for re- sharpening services to Dorn and Hart Microedge. They are located near Chicago, Illinois and their prices are very reasonable. Maybe you can google all three vendors for information if you get in a bind, but I am pretty sure they provide services outside the US. Jack On Apr 21, 2009, at 5:14 AM, yvan lindekens yvan_lindek...@yahoo.com wrote: Hi all, I have some large (25 - 30cm) type B, C and D, both steel and tungsten carbide, microtome knives that need sharpening and/or reconditioning. Does anyone knows a company in Europe (preferably within the EC) that does that kind of work at an affordable price? By the way: what is a *normal* price for those things? Impossible to answer question, I suppose... Thanks in advance! Yvan. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Cloudy MMA embedded block
Ooi, Somehow you are getting moisture/water mixed in with the MMA solution(s). I only remember visiting the IMCB facility, so if this is not your facility then I am not sure of your laboratory environment (air temperature/humidity). The best thing I can tell you at this point is to try and find ways to reduce the chance for moisture accumulation. I am pretty sure that at a minimum you are storing your catalyst and monomer (MMA) at 4C and your softner (DBP) at room temp. Maybe you should take extra steps to ensure your chemicals are at room temp before any use. Lastly, your polymerization is taking too long for your small 20mL vials. My specimens of similar size require only 3-5 days to completely polymerize. Now, I dont remember your concentration of catalyst, but if it is around 2.5g/1L embedding solution, try skipping the 4C step and use room temp (23C) until the specimens are polymerized. Then finish them off in the oven when they are tacky or mostly hard on top. The 4C step is mostly to slow the polymerization for larger specimens requiring a larger volume of solution and/or when the lab temp cannot be controlled routinely at or below 23C. The water bath then also helps to regulate the temperature of the polymerization in the case where the lab environment is too cool (20C) and retarding the rate of polymerzation. The main thing to keep in mind is that the lab environment pretty much controls the process and is probably the main variable that will contrast labs that perform the exact same method. Jack On Apr 21, 2009, at 12:33 AM, ooi.ting.h...@nhc.com.sg wrote: Hi, I am trying very hard to get a clear MMA embedded block. I am appreciate if there is any advice on doing it. I am using 100% MMA (liquid) and 0.5% Perkadox 16 (powder) to do the embedding. I have tried to embed the sample in different conditions. However, it seems to be extremely hard to get a clear embedded block. Below are the methods that I have tried. 1. Before embedding, bring the MMA and perkadox 16 to room temperature. After a few mix it by invertion, I aliquot the solution to the glass vials that contain samples or simply glass vials that without sample. I put the glass vials (both with and without sample) in 4 degree, room temperature and even 37degree oven. 2. Mix the MMA and perkadox when it is cold, invert it a few times. Aliquot it to glass vials that contain sample or without sample. Put in 4degree, room temperature and 37degree oven. I have also tried to speed up the polymerization process by using vacumm for a few samples... Throughout all the sample that I have, I only manage to get a clear empty glass vial which I put in the 4degree for one month and a clear embedded sample which I put in 4 degree overnight and bring it out to room temperature. The rest of the glass vials (with or without sample) are cloudy. I have repeated the methods that I used to get a clear embedded block, I did not get any clear block (for both with and without sample). I am glad if there is any expert advice or guidance. We need a clear embedded sample. Thank you very much! Regards, Ooi _ Confidential information. Unauthorized use or disclosure prohibited. Refer http://www.singhealth.com.sg/ContactUs/#Disclaimer ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] When sectioning small long bones in PMMA ....
I have always orientated the specimen in an AP view and taken longitudinal/frontal sections. As for parallel or perpendicular, I may not be completely sure of what you mean. Jack On Apr 7, 2009, at 9:48 AM, Monfils, Paul pmonf...@lifespan.org wrote: ... such as a mouse femur, do you prefer to orient the bone parallel to the knife edge or perpendicular to the knife edge (or diagonal)? ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Technovit 7200 (isobornylmethacrylate) staining
To my knowledge it is near impossible to obtain an adequate HE stain on thick resin sections. If this is not true, I would be interested in the same information. Jack On Apr 3, 2009, at 6:12 PM, Garcia, Lori, Sr. Scientist lori.gar...@medtronic.com wrote: This question is specifically for Linda Jenkins or anyone who has worked with Technovit 7200, I am trying to do an HE stain on ~70 um thick ground sections on plastic slides. I have followed the Donath method all the way through processing, but the sections do not pick up stains the way the booklet says they should. I am even having trouble following GMA staining procedures that I have found, in fact am afraid to try too many because the acrylic forms tiny cracks that interfere with microscopy. I have researched the histonet archives, but haven't found anything other than a couple of similar inquiries. I would very much appreciate any advice from people who have worked with this specific formulation. Happy Friday! Lori [CONFIDENTIALITY AND PRIVACY NOTICE] Information transmitted by this email is proprietary to Medtronic and is intended for use only by the individual or entity to which it is addressed, and may contain information that is private, privileged, confidential or exempt from disclosure under applicable law. If you are not the intended recipient or it appears that this mail has been forwarded to you without proper authority, you are notified that any use or dissemination of this information in any manner is strictly prohibited. In such cases, please delete this mail from your records. To view this notice in other languages you can either select the following link or manually copy and paste the link into the address bar of a web browser: http://emaildisclaimer.medtronic.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Re: Cortical bone image analysis fluorescent calcein measurement (Damien Laudier)
I will add to Damien's comments and say that another thing to consider is the labeling schedule. Typically the smaller the animal the greater interlabel time period. For example, a smaller window in a mouse tibia/ femur might make it difficult to determine the difference between single and double labels. On the other hand, the remodeling rate can also affect this where in say a larger animal like a dog or sheep with a greater interlabel time period could make you miss your double labels because your first label could be resorbed. Lastly, you are better served with your mouse and rat sections if you can grind your sections thinner at say 20-30 microns. This essentially will sharpen the labels so that a single label can not be mistaken for a double label due to a halo effect from label over-expression. You can accomplish this goal either automatically with say an Exakt grinder or manually with a suction cup and a little patience. Regardless, they are all very teachable methods. I will again encourage you attend the BIOQUANT Image Analysis Meeting April 21-23 in Nashville as all of these topics will be addressed in detail from a histology, histopatholgy, and image analysis perspective. If you can attend, I also encourage you to bring a representative polymerized specimen and/or prepared slides. Jack On Mar 28, 2009, at 10:23 PM, Damien dml...@gmail.com wrote: Hi Jamie, Goal 1: For fluorescent label measurements, you’re definitely better off mea suring multiple fields using a higher power objective (at least10X). Accuracy is paramount when measuring inter-label width and working at a higher magnification will help to ensure this. Your other option is to create a photo-stitched composite image of each sample (you would take multiple images until you get the entire circumference of the sample) and make measurements from a static image. Osteometrics *Osteomeasure* software is not “overkill” and is considered the simpler of the two popular bo ne histomorphometry packages on the market. The beauty of using such programs specifically designed for bone histomorphometry is that the calibration standards/code are already established/programmed, as well as the back-end calculations required to derive MAR. You can certainly make these measurements using Zeiss axiovision software or any imaging software (NIH Image J, Matlab IPT). However, you’ll need to establish your own par ameters and this will require more derivative calculations on your part after principle data collection; a great option if you’re comfortable doin g this. Goal 2: If you’re using your (expensive) precision saw correctly, you should not be getting uneven sections. Try reducing your cutting speed and/or adjusting the angle of your sample. Also, make sure you’re using a blade of th e proper size- relative to the size of the sample. Use a micrometer to gauge your cutting adjustments. Once you fix this problem, yes it is possible on a 1mm section, but since all confocal microscopes are not created equally, success will ultimately depend on the resolving power of your system. Good luck! -Damien Laudier ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Plastic Thin Sections Won't Stay
Try coating your slides with Haupt's adhesive. It can be purchased commercially from Fisher (or you can make it yourself using glycerin, gelatin, and phenol) and then cut it 1:1 with 50% EtOH to prepare a working dilution. I have used Haupt's for many years on both small (1x3 slides) and large (2x3 slides) and have NEVER lost a section (knocking on wood now...LOL) to staining. Make sure to look over your slides before use to insure adequate coating of the slide. Please keep in mind that with every new thing you bring into the lab, you should do some testing of your own. Specifically, you will want to research the concentrate that works best for your staining as it does yield to some degree of background when staining with hematoxylin, fast green, and aniline blue if the concentration is too high. Jack Ratliff Date: Wed, 25 Mar 2009 15:58:05 -0400 From: har...@medinst.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Plastic Thin Sections Won't Stay We are re-visiting thin sectioning of MMA embedded items. The sections are great, but none of them stay on the slides for the staining. We're trying Sta-On, and have tried chrome alum slides. We use a butoxy mixture to stretch them and immuno quality slides. Any ideas? Thanks for your help! -- Corliss Harris Histology Technician MED Institute 1 Geddes Way West Lafayette, IN 47906 765-463-7537 ext. 1150 765-497-0641-fax ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] (no subject)
Ooi, As you know, working with MMA is tricky enough to have to worry about losing sections during staining. Unfortunately, I cannot speak with experience in using Bond-rite slides, but maybe there is someone monitoring this message thread that can provide helpful information on that topic. However, I can speak to the experience of using Haupts coated slides. First, it is not uncommon to observe hematoxylin background staining from Haupts coated slides. Second, you are correct in that it takes just the right concentration of Haupts solution in order to retain section adherence to the slide. Given these two observations, allow me to provide a few suggestions for working with Haupts coated slides. Before I prepare my slides, it is necessary to prepare a working dilution of Haupts solution. Whether or not you use in house prepared or commercially prepared concentrate, you should typically look to a ratio of 1:1 with liquified concentrate to 50% EtOH. This is a pretty standard dilution that works for me. You definitely do not want to go higher than a 50% concentration of Haupts, but you could try tweaking it a little within the 40-50% range. I would suggest that maybe you do a short experiment using different dilutions of the Haupts concentrate so that you may zero in on that one dilution that will minimize background staining and optimize section adherence. You may not have perfection with the hematoxylin background or aniline blue in a trichrome stain (I usually substitue aniline blue with SF light green yellowish), but you may find something that you can live with for the HE. Next, you might then try to work with the hematoxylin staining a bit. Specifically, maybe you can tweak the hematoxylin staining time by keeping it standard (I use hematoxylin 2 from Richard Allen and typically stain for 3 minutes) and/or increasing it a bit so that you can extend the acid clarifier step (I clarify or decolorize for 20 seconds) a little longer to help wash out or decolorize the background staining. Obviously you do not want to compromise your nuclear detail, but this should help a bit. Please feel free to ask any additional questions as needed. Jack Ratliff Date: Wed, 18 Mar 2009 05:33:01 -0700 From: ooitingh...@yahoo.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] (no subject) Hi, I just began to work with the methy methacrylate embedding plastic section. I am facing the difficulities to attach the sample on the glass slide. I have tried using Haupts adhesive coated slides and also the bond-rite slide to attach my 5micron sample. I am doing normal hematoxylin and eosin staining. The section always detach from the glass slide towards the end of the staining. I need to use the concentrated Haupts adhesive coated slide to reduce the chance for the section from lift up from the glass slide. However, I observed the high and dirty background after the staining. I noticed that some users are using Bond-rite slides to attach their samples. I tried this. However, the sections always lifted up towards the end of the staining. I am not too sure whether is my technique that giving me this kind of problem. I prewarmed the Bond-rite slides and put my sections in the 55C waterbath. Then I fished the section using the Bond-rite slide and lay it down on 55C heater. And finally in the oven incubator for more than 18 hours at 55C before staining... I would appreciate if there is any suggestion that can help to attach the section on the glass slide. Thank you very much! Yours sincerely, Ooi Singapore ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Von Kossa staining on PMMA sections
Paul, For MMA embedded specimens (MMA +DBP), I first deplastify my sections, hydrate to water, stain in 5% silver nitrate solution for 5 minutes (in the dark), wash times three changes in DI H2O (in the dark), develop in sodium-carbonate formaldehyde solution for 2 minutes (in the dark), wash times two in DI H2O (back under normal lighting conditions), then stop the reaction in sodium thiosulfate + potassium ferricyanide solution for 30 seconds, and immediately rinse in running tap water for 15 minutes. The Von Kossa reaction results from process above then yields black mineralized bone. After the tap water rinse, I generally counterstain with 2% MacNeal's tetrachrome for 5 minutes, rinse in DI H2O, and dehydrate to xylenes to coverslip. This then reveals immature bone formation or osteoid = grayish or jaded green, growth plate cartilage = purple, osteoblasts = blue, osteoclasts = blue-green, bloods cells = greenish, etc. Feel free to contact me if you have any additional questions. Jack Date: Thu, 12 Mar 2009 11:19:49 -0400 From: pmonf...@lifespan.org To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Von Kossa staining on PMMA sections The standard Von Kossa silver stain for calcium calls for 20 minutes in the silver nitrate solution under UV light. There is a modified Von Kossa for plastic embedded bone sections, which is identical except it calls for a minimum of 6 hours in the silver nitrate solution under UV. Does anyone know why such a long staining time is recommended? Visually the calcium in the bone sections turns black within 20 minutes, so why is so much additional time needed? Thanks. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
