[Histonet] TRAP Stain Help
Hey guys, recently I've used the Sigma Aldrich TRAP Stain Kit in order to stain for Osteoclasts in Formalin Fixed Paraffin Embedded bone tissues that are EDTA decalcified. Unfortunately there was no TRAP stain to be found whatsoever on all slides that I stained. I was hoping that someone with some experience with TRAP stain could really help me out! Here are a couple of reasons that I have wondered as to why the TRAP stain might not be visible 1. Staining too long with Hematoxylin counterstain? I have noticed in several trial runs that sometimes if the Hematoxylin counterstain is too long then this can effect the amount of TRAP stain that shows up with the osteoclasts. Perhaps I'm just over analyzing 2. Perhaps no osteoclasts were present to even staindoes anyone know what the usual ratio of Osteoclasts to Osteocytes are? In the past stains that I have usedwhenever there is an osteoclasts that shows up...it is usually pretty spotty and many times they are in groups together...is this how it normally is? 3. Perhaps the TRAP stain is being washed away through the rinsing with dI or dehydration with ethanol/clearing just before mounting. A protocol that I used before the Sigma Aldrich Kit incorporates incubating the slides in 37 celsius water for 1 hour just prior to allowing the slides to sit in the TRAP stain solution (this is also 37 celsius in the same water bath) and letting it sit for 20 minutes. With this stain...there seems to be a consistent showing of osteoclasts but I'm just not sure if all of the osteoclasts are showing up correctly...that is why I moved to the Sigma Aldrich kit to make sure and the results didn't show ANY osteoclasts there! Anyways...I'm just curious if there are any reasons that the TRAP stain is not showing up...I would appreciate any input! Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TRAP Stain Troubleshooting
Hello Histonetters! I have a few questions regarding TRAP Staining and I'm hoping someone who has experience with this stain could help me out. I have been using a protocol that is apparently not goo because it hasn't shown any TRAP whatsoever. I have seen various themes in several sources, one of those is that slides are incubated for 1 hour in pH 9.4 TBS before being put into the TRAP staining solution. My question for this is why? In my protocol, I have a TRAP incubation medium that contains the tartaric acid, that is at pH of 5.0, which I then combine with Naphthol and Fast Red (which are the reagents responsible for the chromogenicity of the stain). I guess I'm confused as to what the tartaric acid is used for as wellwhich I'm speculating that it denatures most of the enzymes other than TRAP (hence the name Tartrate Resistant Acid Phosphatase). Therefore, I see the three main steps for this protocol to be: 1. Incubate in TBS pH 9.4 (which I'm still confused why) 2. Combine with Tartaric Acid to denature all of the other enzymes to localize osteoclasts 3. Add Naphthol and Fast Red to Identify where TRAP is. Therefore, I'm confused why I am combining Tartaric Acid with my Napthol and Fast Redis this a common method that is done to eliminate multiple unnecessary steps? If somebody has a protocol that works consistently for them and wouldn't mind sharing that with me, I would very appreciative of you! I'm just trying to understand this stain so that I can somehow tweak it to work. Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TRAP staining protocol troubleshooting
Hello histonetter heroes! I have an issue with a TRAP staining protocol that I'm currently using that involves Fast Red Violet LB Salt for the staining of the Osteoclast and then Fast Green as a counterstain. The osteoclast are suppose to stain Red Violet and the background is suppose to stain green. However, I'm not seeing any Red Violet Osteoclast but just really really Green tissue. The bone I was using was cortical bone but surely there would at least 1 Osteoclast in all of sections that I had. Here is the protocol that I'm using: Trap Staining solution Mix: Trap Basic Incubation medium, Fast Red Violet LB Salt, and Naphthol AS-MX Phophaget substrate mix Procedure: 1. Place TRAP staining solution mix in staining dish and pre-warm to 37 degree celsius in waterbath 2. Deparafinnize slides and rehydrate through graded ethanols to distilled water. 3. Place slides in pre-warmed TRAP staining solution mix and incubate at 37 celsius for 30 mins or until control is developed. 4. Rinse in distilled wtaer 5. Counterstain with 0.02% Fast Green for 30 seconds and rinse quickly in distilled water. 6. Dehydrate quickly thorugh graded alcohols, 5 seconds each, clear in Xylene and mount. The problem is that the main staining with the Fast Red is not very substantial.at least not as much as it should be. When I take the slide from the water bath, the tissue section is only slightly red...and may even look more orangish yellowwhich is a long ways away from red violet. Then when I counterstain with Fast Green, I feel that the Fast Green stain is so prominent that it just superimposes the Fast Red stain. For anyone that has experience with TRAP staining or Fast Red Violet staining then that would be awesome to hear your input! 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] Decalcified Bone Clearing time
Hey everyone! I'm having some issues about clearing the bone tissues after they get done dehydrating from decalcification. I know that over clearing the bone tissues in Xylene for too long can cause them to become very brittle and I'm afraid that that was what was happening. The bone blocks are not very bigtheir weights are listed as 0.222g, 0.100g, and 0.052g. The biggest bone block is about 4mmx4mmx4mm so it's not very big I would say. I cleared with 50% Ethanol/50% Xylene for 1 hour, and then I changed it with 100% Xylene for another hour. The bone blocks become clear as expected but they begin to get a yellowish-tan tinge to them. I'm not the most experienced but from what I've studied, I don't think that 2 hours in Xylene is overkill by any means so maybe it's something that I'm doing subconsciously.if anyone has any suggestions are ideas please let me know! :) 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
Re: [Histonet] TRAP staining protocol troubleshooting
That's interesting because I buffered it to 4.7-5.0..that's actually a huge difference.does that have to do with he Fast Red approach? Sent from my iPhone On Nov 17, 2014, at 2:40 PM, Patsy Ruegg prueg...@hotmail.commailto:prueg...@hotmail.com wrote: Actually trap will work if you pre incubate it in 0.2M Tris buffer Ph9 (use sodium hydroxide to pH) for 1 hour at 37dc then proceed with the trap stain without rinsing. Check the stain in 20 min as it develops quickly after this. This protocol was given to me by the late great Hermina and it works really well even on formic acid decaled FFPE bone in my hands. Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.commailto:prueg...@hotmail.com pru...@ihctech.netmailto:pru...@ihctech.net From: prueg...@hotmail.commailto:prueg...@hotmail.com To: wa...@livemail.uthscsa.edumailto:wa...@livemail.uthscsa.edu Subject: RE: [Histonet] TRAP staining protocol troubleshooting Date: Mon, 17 Nov 2014 13:22:24 -0700 Trap dose not work on decalcified bone unless you have decaled with EDTA in my experience. Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.commailto:prueg...@hotmail.com pru...@ihctech.netmailto:pru...@ihctech.net From: wa...@livemail.uthscsa.edumailto:wa...@livemail.uthscsa.edu To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Date: Mon, 17 Nov 2014 18:58:50 + Subject: [Histonet] TRAP staining protocol troubleshooting Hello histonetter heroes! I have an issue with a TRAP staining protocol that I'm currently using that involves Fast Red Violet LB Salt for the staining of the Osteoclast and then Fast Green as a counterstain. The osteoclast are suppose to stain Red Violet and the background is suppose to stain green. However, I'm not seeing any Red Violet Osteoclast but just really really Green tissue. The bone I was using was cortical bone but surely there would at least 1 Osteoclast in all of sections that I had. Here is the protocol that I'm using: Trap Staining solution Mix: Trap Basic Incubation medium, Fast Red Violet LB Salt, and Naphthol AS-MX Phophaget substrate mix Procedure: 1. Place TRAP staining solution mix in staining dish and pre-warm to 37 degree celsius in waterbath 2. Deparafinnize slides and rehydrate through graded ethanols to distilled water. 3. Place slides in pre-warmed TRAP staining solution mix and incubate at 37 celsius for 30 mins or until control is developed. 4. Rinse in distilled wtaer 5. Counterstain with 0.02% Fast Green for 30 seconds and rinse quickly in distilled water. 6. Dehydrate quickly thorugh graded alcohols, 5 seconds each, clear in Xylene and mount. The problem is that the main staining with the Fast Red is not very substantial.at least not as much as it should be. When I take the slide from the water bath, the tissue section is only slightly red...and may even look more orangish yellowwhich is a long ways away from red violet. Then when I counterstain with Fast Green, I feel that the Fast Green stain is so prominent that it just superimposes the Fast Red stain. For anyone that has experience with TRAP staining or Fast Red Violet staining then that would be awesome to hear your input! 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.edumailto: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] Decalcified Bone Staining Problems
Hey guys! I was curious if anyone could send me a link or maybe even just a source where I could see the difference between a properly/completely decalcified HE stained bone tissue vs. an improper/incompletely stained decalcified specimen. This would be beneficial because I believe that the tissues that I am currently staining with HE are not totally decalcifed, therefore there are sections in the bone tissue that are stained a bit more heavily than others...and this pattern of stains holds pretty consistently with the all of the sections that were taken from one specific bone block. In short, I would just love to see what a quality stained uniform HE section looked like. Here are several possible explanations as to why my slides are not staining uniformly: - Perhaps the way I am letting the slides dry? - when the slides are completely done staining and clearing, I let them dry and perhaps that is something to do with it. - I am not putting coverslips on the slides yet because I just want to get the staining protocol down first...perhaps if I put the coverslips on then that would help solve the problem? 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] Re: Decalcified Bone Eosin Overstaining
Wow these are awesome, thanks everybody for feedback! So appreciated Sent from my iPhone On Oct 2, 2014, at 7:05 PM, Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au wrote: The increased eosinophilia, in this scenario, is usually due to over-decalcification. I would suggest: *Treat sections with 1% Periodic acid (as used in the PAS stain) for 10 minutes before Haematoxylin staining. *Use an iron-haematoxylin or Celestine blue-Haematoxylin *decrease time in eosin (or use a neutral 1% eosin stain). This should increase nuclear staining and decrease the eosin reaction. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist, the Children's Hospital at Westmead Adjunct Fellow, School of Medicine, University of Western Sydney Tel: 612 9845 3306 Fax: 612 9845 3318 Pathology Department the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Wait, Trevor Jordan Sent: Friday, 3 October 2014 4:11 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Decalcified Bone Eosin Overstaining Hey histonetters! I'm currently doing some HE Staining for EDTA Decalcified Bone Tissue and it seems that the tissue is staining very intensely with Eosin...perhaps too much. Was curious to see from some of the more experienced histotechnicians out there which methods you guys have used in the past to perhaps fix an issue like this. Here is my protocol that I have used once I have sectioned the paraffin embedded tissue. I have omitted the 1 second Acid Alcohol Dip in a few of my trials and it seemed that this method worked great to keep the tissue from overstaining with Eosin. However, this is an important step for differentiation of the Hematoxylin...so I'm a little puzzled about what to do next. Perhaps wash with tap water again after the Acid Alcohol? 1. Xylene - 3 Minutes 2. Xylene 3- Minutes 3. Xylene - 3 Minutes 4. 100% EtOH - 3 Minutes 5. 100% EtOH - 3 Minutes 6. 95% EtOH - 3 Minutes 7. 70 % EtOH - 3 Minutes 8. Filtered Harris Hematoxylin (Commercial Purchase) - 6 Minutes 9. Wash with running Tap water 10. 1 second dip in 0.5% 12M HCL Acid Alcohol 11. Eosin Y Solution - 1 Minute 12. 70% EtOH - 2 Minutes 13. 70% EtOH - 2 Minutes 14. 95% EtOH - 2 Minutes 15. 95% EtOH - 2 Minutes 16. 100% EtOH - 2 Minutes 17. 100% EtOH - 2 Minutes 18. Xylene - 1 Minute 19. Xylene - 1 Minute 20. Xylene - 1 Minute 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 * 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 are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Sydney Children's Hospitals Network. This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Sydney Childrens Hospital's Network accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Decalcified Bone Eosin Overstaining
Hey histonetters! I'm currently doing some HE Staining for EDTA Decalcified Bone Tissue and it seems that the tissue is staining very intensely with Eosin...perhaps too much. Was curious to see from some of the more experienced histotechnicians out there which methods you guys have used in the past to perhaps fix an issue like this. Here is my protocol that I have used once I have sectioned the paraffin embedded tissue. I have omitted the 1 second Acid Alcohol Dip in a few of my trials and it seemed that this method worked great to keep the tissue from overstaining with Eosin. However, this is an important step for differentiation of the Hematoxylin...so I'm a little puzzled about what to do next. Perhaps wash with tap water again after the Acid Alcohol? 1. Xylene - 3 Minutes 2. Xylene 3- Minutes 3. Xylene - 3 Minutes 4. 100% EtOH - 3 Minutes 5. 100% EtOH - 3 Minutes 6. 95% EtOH - 3 Minutes 7. 70 % EtOH - 3 Minutes 8. Filtered Harris Hematoxylin (Commercial Purchase) - 6 Minutes 9. Wash with running Tap water 10. 1 second dip in 0.5% 12M HCL Acid Alcohol 11. Eosin Y Solution - 1 Minute 12. 70% EtOH - 2 Minutes 13. 70% EtOH - 2 Minutes 14. 95% EtOH - 2 Minutes 15. 95% EtOH - 2 Minutes 16. 100% EtOH - 2 Minutes 17. 100% EtOH - 2 Minutes 18. Xylene - 1 Minute 19. Xylene - 1 Minute 20. Xylene - 1 Minute 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] Weight Loss/Weight Gain Decal
Hello all! I'm currently doing some decalcification and was curious if anyone had some particular advice about the weight loss/weight gain method. I understand that when the decalcification process is complete, the tissue block will begin to increase in weight. However, I'm confused when I should record the weight for the block once they have been taken out of the EDTA solution. You see, for the times I weighed the blocks before... the weights were a little skewed because there were differing amounts of solution on the blocks while they were sitting on the balance. I just want to standardize my protocol a little more so that I can be sure the block is actually gaining weight due to the calcium loss rather than just extra solution sitting on the outside of the block. Would letting the blocks sit out of solution for about 30 minutes before being weighed help with the matter? I know that the blocks take on water once they are completely decalcified so I'm not sure how much this will affect that. 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
RE: [Histonet] Weight Loss/Weight Gain Decal
Ha Wow...that's almost too easy. Thank you for this! 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 From: Jennifer MacDonald jmacdon...@mtsac.edu Sent: Monday, August 11, 2014 2:33 PM To: Wait, Trevor Jordan Cc: histonet@lists.utsouthwestern.edu; histonet-boun...@lists.utsouthwestern.edu Subject: Re: [Histonet] Weight Loss/Weight Gain Decal I believe this was originally from Patsy Ruegg Decalcification End Point: Weight Loss, Weight Gain 1.Blot sample to remove excess fixative 2.Weigh bone in mg, record as beginning weight 3.Next day, rinse bone, blot and weigh bone daily, record weight. Change decalcifying solution to refresh acid OR EDTA. Return bone to fresh decalcifying solution. 4.When bone begins to GAIN weight, remove from decalcifying solution, rinse and process. YOU MUST WEIGH IN MILLIGRAMS FOR ACCURACY Once calcium is totally removed (bone loses weight as this happens), water replaces the calcium and weight begins to go up. This is the point at which calcium should be totally gone. The original method used a chemical test at the end to insure no calcium was in the decalcifying solution. If you do this, you cannot stir the solution during decalcification. Be sure to suspend bone in the solution to insure all sides of bone are in contact with decalcification solution. From:Wait, Trevor Jordan wa...@livemail.uthscsa.edu To:histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date:08/11/2014 12:29 PM Subject:[Histonet] Weight Loss/Weight Gain Decal Sent by:histonet-boun...@lists.utsouthwestern.edu Hello all! I'm currently doing some decalcification and was curious if anyone had some particular advice about the weight loss/weight gain method. I understand that when the decalcification process is complete, the tissue block will begin to increase in weight. However, I'm confused when I should record the weight for the block once they have been taken out of the EDTA solution. You see, for the times I weighed the blocks before... the weights were a little skewed because there were differing amounts of solution on the blocks while they were sitting on the balance. I just want to standardize my protocol a little more so that I can be sure the block is actually gaining weight due to the calcium loss rather than just extra solution sitting on the outside of the block. Would letting the blocks sit out of solution for about 30 minutes before being weighed help with the matter? I know that the blocks take on water once they are completely decalcified so I'm not sure how much this will affect that. 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
[Histonet] Low vs. High Profile Blades
Hey does anyone know the difference between a High Profile Blade vs. a Low Profile Blade when using for sectioning of Paraffin embedded tissues specimens? Which one would ya'll prefer for decalcified bone? 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
RE: [Histonet] Bone Histology Protocol
Thanks so much for the reference and I will visit that site and use that site most definitely as a resource. However, the reason I am wanting to decalcify is that my researcher (my employer) has instructed me that that is the way we will be doing it lol. I have read about resin/plastic embedding and that method does seem pretty efficient to use so I'm not sure why he opted to not go the plastic resin route. I do know that we have a limited budget to work with so maybe this is the cheapest route we can afford, along with a microtome that might not be sufficient to cut through hard bone and plastic. As far as the species of bone that we are processing, that too is something I'm not exactly sure about, but is something I should probably find out because from my readings, this is an important aspect. 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 From: Jack Ratliff ratliffj...@hotmail.com Sent: Saturday, March 1, 2014 3:16 AM To: Wait, Trevor Jordan Cc: histonet@lists.utsouthwestern.edu; Jack Ratliff Subject: 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] Bone Histology Protocol
I'm terribly sorry, when you asked about species...my inclination was that you were curious which type of bone we were using (femur, humerus, tibia, etc. and cortical/cancellous) but we will be using human bone for this research project that has been donated from various orthopedic surgeries. 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 From: Jack Ratliff ratliffj...@hotmail.com Sent: Saturday, March 1, 2014 3:16 AM To: Wait, Trevor Jordan Cc: histonet@lists.utsouthwestern.edu; Jack Ratliff Subject: 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
[Histonet] RE: Bone histotechniques
Thank you so much for the materials, they were definitely helpful for the EDTA and I very much appreciate that! Yes I have contacted my Researcher, unfortunately it is the weekend whenever I started to research for this protocol so my answers may not come til Monday morning. However, I do know that our species is human bones that have been donated from various orthopedic surgeries. As far as the exact type of bone (tibia, femur, etc.) I do not know the answer for they are just shavings or scraps that have been conserved from the surgeries that would otherwise be exposed of. I will also obtain that information as well. 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 From: gayle callis gayle.cal...@bresnan.net Sent: Saturday, March 1, 2014 10:13 AM To: Wait, Trevor Jordan Subject: Bone histotechniques Trevor, Please say what kind of bone you are working with, including species, femur, tibia, etc. Attached is an excellent EDTA decalcification that was developed years ago by a bone expert/professor, and is published. Once you give more bone details, then I can expound on how to do this. You will probably get many replies. Also, are you using automated tissue processing or have to do this by hand? What are you to do for staining? Immunohistochemistry? Other staining only? There are a lot of variables here that affect how you fix, etc for the bone you are working with. Gayle Callis HTL/HT/MT(ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Bone Histology Protocol
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
