RE: [Histonet] What is a great manual mirotome
I paid $30.00 for my pre-owned Spencer 820. Liked it so well I bought two. It has served as a virtually foolproof and indestructible workhorse for 22 years. I have produced, literally more than a tone of slides with it. Jerry L Ricks Research Scientist University of Washington Department of Pathology Date: Tue, 12 Jul 2011 12:57:28 -0700 From: vic...@pathology.washington.edu To: b-freder...@northwestern.edu Subject: Re: [Histonet] What is a great manual mirotome CC: Histonet@lists.utsouthwestern.edu The old black ones, gotta love them. Most of the young techs out there probably have never seen one. Victor Tobias HT(ASCP) Clinical Applications Analyst University of Washington Medical Center Dept of Pathology Room BB220 1959 NE Pacific Seattle, WA 98195 vic...@pathology.washington.edu 206-744-2735 206-744-8240 Fax = Privileged, confidential or patient identifiable information may be contained in this message. This information is meant only for the use of the intended recipients. If you are not the intended recipient, or if the message has been addressed to you in error, do not read, disclose, reproduce, distribute, disseminate or otherwise use this transmission. Instead, please notify the sender by reply e-mail, and then destroy all copies of the message and any attachments. On 7/12/2011 12:46 PM, Bernice Frederick wrote: I want my AO820 back! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patrick Laurie Sent: Tuesday, July 12, 2011 2:40 PM To: sdys...@mirnarx.com Cc: Histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] What is a great manual mirotome I've always been a fan of the Leica microtomes, I've used all kinds, from the Leica RM 2025 Microtome to the Leica RM 2265. A good non-automated version is the RM 2235. I've also used the Microm (thermo-fisher-shandon) HM325S which work well. The Sakura Accucut SRM is manufactured by Leica, I believe it is equivalent to the RM2025. I however have never used the Leica 2125. I've always had good service from them, especially if you have a service contract. Good luck! On Tue, Jul 12, 2011 at 12:11 PM,sdys...@mirnarx.com wrote: I am a manual tome junky! I like the Thermo Finesse (I think it's 325). These are the old microms...they work like a charm =) Sarah Goebel-Dysart, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of lsc...@sfcn.org Sent: Tuesday, July 12, 2011 1:22 PM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] What is a great manual mirotome Hi, Our small lab is looking for some advise on what microtome to replace a fairly new Leica RM2235 with. We have been looking at the Sakura SRM 200 and the Leica 2125. We are looking for reliability and have net gotten it from the RM2235. Is anyone using either of these that would be willing to offer suggestions? Thanks, Scott Hendricksen HT (ASCP) ___ 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 -- Patrick Laurie HT(ASCP)QIHC CellNetix Pathology Laboratories 1124 Columbia Street, Suite 200 Seattle, WA 98104 plau...@cellnetix.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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Vasculature in tumors
You are probably looking for capillaries maybe arterioles so there wont be any elastic lamina for the pentachrome stain to detect. Instead try IHC with an endothelial marker like VE Cadherin (best) or in a pinch, CD31 or PECAM. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Fri, 1 Jul 2011 12:48:33 -0500 From: sgoe...@mirnarx.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Vasculature in tumors Hello all and happy long weekend (yes our boss gave us the rest of the day off at 2!!!) Looking to stain vasculature in tumors. I did trichrome, pentachrome, and PAS (with digestion) and am being told that these are not optimal for what they are looking for. Any other ideas to stain blood vessels would be awesome!! Thanks ya'll! Sarah Goebel-Dysart, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ 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] Keeping Histo room floor clean?
We keep getting a lot of paraffin on the floor of one histo room--especially around the microtome and the embedding station. Short of laying down a tarp, what do folks do keep wax off of the floor? Thanks, Jerry Ricks Research Scientist University of Washington Department of Pathology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] why no replay??--Alcian Blue
According to Dako, At pH 2.5, Alcian Blue stains sulphated and non-sulphated acidic carbohydrates. At pH 1.0, only sulphated carbohydrates are stained. Jerry RicksResearch Scientist University of WashingtonDepartment of Pathology Date: Fri, 3 Jun 2011 12:21:31 -0700 From: k8...@yahoo.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] why no replay?? i have posted my quistion 2-3 times several days ago and recieve no negative or positive comment !!! why?? is my quistion not clear or what?? --- On Thu, 6/2/11, mohamed abd el razik k8...@yahoo.com wrote: From: mohamed abd el razik k8...@yahoo.com Subject: Alcian blue quistion To: histonet@lists.utsouthwestern.edu Date: Thursday, June 2, 2011, 10:53 AM hi every body i have a confusing quistion reagrding Alcian blue pH 1 2.5 stains. suppose you have 100 goblet cell react positive to pH 1 which mean all have sulfated mucin. and 50 cell from other section react positive to pH 2.5 which mean acidic type(sulfated or sialated). why these 50 cell only react to pH 2.5 althogh the other non reacted cells possess acidic sulfated type?? on other word i need to know for what type of mucins alcian B 2.5 react with? thanx all ___ 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] Alternatives to Cytospin?
I will be collecting bronchoalveolar lavage fluid (BALF) from mice and want to do do a diff-quick stain on the fluid to characterize the leukocyte population. My colleagues usually use a cytospin to concentrate cells onto a slide but I'll be at a lab that doesn't have the equipment so I'm looking for alternatives. Could I just put a drop of the BALF onto a charged slide and air dry it? Or treat the BALF like a blood smear? Alternatively, could I fix the cells in...ethanol maybe and then cytospin them the next day when I get back to my lab? Any ideas will be much appreciated. Jerry Ricks Research Scientist University of Washington Department of Pathology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Counter top Immunos...
For mouse and rabbit primary antibodies I like the Vector ImmPRESS kits. They save time. For HRP substrates Nova Red looks too dull to me. I have had good results with AEC and also with Vector ImmPACT DAB. Jerry Ricks Research Scientist University of Washington Department of Pathology -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Marshall, Kimberly K Sent: Thursday, June 02, 2011 9:57 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Counter top immunos Hello Histo folks Hope everyone is having a great weekI am writing to get some info on immuno kits for staining just by hand, like back when I first started in this field. We are a small lab in Alaska and all of our immunos are sent out of state, We are looking to do some of them here and try to save money, but don't really need a stainer. So are there any kits that are better than others? Is there anyone that does immunos this way that would give some much needed advise??? Again thanks in advance for all the imput... Kimberly ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Recycling slide brite
Looks like B/R has a gadget that will do it... http://www.solvent--recycling.com/xylene_substitutes.html Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Tue, 26 Apr 2011 12:51:15 -0700 From: rjbu...@yahoo.com To: Histonet@lists.utsouthwestern.edu; dwenze...@gmail.com Subject: Re: [Histonet] Recycling slide brite CC: Being a MIXTURE of hydrocarbons, it will crack at different temperatures (separately). You would have to reconstitute it using the same proportions (usually proprietary). René J. --- On Tue, 4/26/11, Dawn Herron dwenze...@gmail.com wrote: From: Dawn Herron dwenze...@gmail.com Subject: [Histonet] Recycling slide brite To: Histonet@lists.utsouthwestern.edu Date: Tuesday, April 26, 2011, 2:25 PM Hello histonetters. We have made the transition from xylene to using slide brite in our stainer and for coverslipping and have worked out all the kinks except one---recycling it successfully. We've been talking to our recycler manufacturer (BR) and the manufacturer of slide brite and still haven't had a successful run. Does anyone out there use this product and recycle it? If so could you send me your recycling protocol? Any thoughts would be appreciated. Thanks, Dawn ___ 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] Supplier for human testis control slides?
I'm working up a new antibody and human testis is the suggested postive control. Does anyone know of a supplier? Thanks, Jerry Ricks Research Scientist University of Washington Department of Pathology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Rat heart valve histology trimming and sectioning reg
Assuming you are talking about the aortic valve Remove the heart and dissect away as much surrounding fat as you can. Leave some aorta attached. Make sure the heart is very well fixed in formalin. If the heart is soft and floppy rather than firm the next step wont go well. The next step is to remove most of the ventricles--you don't want to section through all that and it just gets in the way of proper orientation during embedding. There are two ways to trim off the ventricle. The critical thing is that your sectioning plane is perpendicular to the angle at which the aorta exits the heart. The classic way is to lay the heart in the anatomical position and use a straight blade, like a razor, to cut through it, guillotine-fashion along a line drawn between the lower margins of the atria and which is perpendicular to the angle at which the aorta exits the heart. I found that method to be not so reproducible so I came up with another way. Under a dissecting scope, I use my right hand and fine forceps to suspend the heart by the stub of the aorta. Gravity then automatically pulls the heart into almost the right position for the next step. Next I use my left hand and a straight back forceps to grasp the heart just below the atria. Then trim the aorta very close to the heart. Use your fine forceps to twiddle the angle that the heart sits in the straight forceps until you are looking directly down into the aortic sinus. You should be able to see the valves. Now take a number eleven scalpel and in one or two smooth strokes, run it along the edge of the straight forceps, separating the ventricles from the top of the heart. If you do it right you will have a sort of loaf shaped piece of tissue. If you lay it flat under the scope, the stub of the aorta will be pointing straight up and you will be able to see the valves. Embed the tissue so that the caudal side is at the bottom of the embedding mold. Throw away sections until you start to see the valves. Until you have cut a few and know what you are doing it is best to start saving sections early rather than later. I like to put three sections per slide and initially stain slides 1,3,4,7,9...etc, saving the rest for possible IHC. My prefered stain for lesions is the Modified Movat Pentachrome. It's kind of a pain to do but for lesion composition analysis it can't be beat. I start lesion analysis at the point where I can see all the leaflets. Stop when only the valve stems are visible. Anyway, it will take you a while before you get reproducible, nice cross sections. That first trimming away of the ventricles makes it or breaks it. Good luck and feel free to ask clarifying questions. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Fri, 17 Sep 2010 08:39:40 + To: histonet@lists.utsouthwestern.edu From: abija...@rediffmail.com Subject: [Histonet] Rat heart valve histology trimming and sectioning reg Dear Histonetters, Our pathologists are interested in rat heart valvular lesions. I am working on trimming and sectioning methods to get uniform and reproducible morphology of heart valves in paraffin sections. It seems that available literature dealing with this is limited. Requesting your valuable experience in this regard. Thanks a lot Abi jagannath ___ 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: Formalin and Xylene odor containment
A fume hood is the traditional, legal and effective technology. From: janice.maho...@alegent.org To: del...@musc.edu; jphistol...@gmail.com; histonet@lists.utsouthwestern.edu Date: Fri, 9 Jul 2010 14:14:02 -0500 Subject: RE: [Histonet] Formalin and Xylene odor containment CC: Great advice Vinnie, At a prior job our safety manager found there to be more problems with ozone than what we were trying to fix. Jan mahoney Omaha, NE -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Della Speranza, Vinnie Sent: Friday, July 09, 2010 2:09 PM To: 'Joao Pessoa'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Formalin and Xylene odor containment I don't have any expertise in this area however I recall reading about the controversies of ozone generators. You would do well to do a bit of internet research before you take this leap. If your facility has a safety officer I'd definitely determine if there is an institution position on ozone generators. Here is one link for information but you shouldn't have difficulty coming up with others. http://www.epa.gov/iaq/pubs/ozonegen.html Vinnie Della Speranza Manager for Anatomic Pathology Services Medical University of South Carolina 165 Ashley Avenue Suite 309 Charleston, South Carolina 29425 Tel: (843) 792-6353 Fax: (843) 792-8974 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joao Pessoa Sent: Friday, July 09, 2010 3:01 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formalin and Xylene odor containment All, We have recently been using a new piece of demo equipment to control formalin, xylene and other odors. The unit is an air purifier from BioZone Scientific. Our ventilation is good in all areas of our lab and we have always been found to be well below the particulate count allotment for xylene, formalin, and other compounds. However, many area of the lab still had a heavy chemical smell (like a beauty salon, they say). This smell goes away with the BioZone unit, which uses UV light and ozone to break down the organic compounds. Does anyone else has experience with BioZone? We are thinking about buying it, but I wanted to hear the opinion of others. Cheers, Joao Histo Tech ___ 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 Sponsored by Catholic Health Initiatives and Immanuel, Alegent Health is faithful to the healing ministry of Jesus Christ, providing high quality care for the body, mind and spirit of every person. The information contained in this communication, including attachments, is confidential and private and intended only for the use of the addressees. Unauthorized use, disclosure, distribution or copying is strictly prohibited and may be unlawful. If you received this communication in error, please inform us of the erroneous delivery by return e-mail message from your computer. Additionally, although all attachments have been scanned at the source for viruses, the recipient should check any attachments for the presence of viruses before opening. Alegent Health accepts no liability for any damage caused by any virus transmitted by this e-mail. Thank you for your cooperation. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ The New Busy think 9 to 5 is a cute idea. Combine multiple calendars with Hotmail. http://www.windowslive.com/campaign/thenewbusy?tile=multicalendarocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_5___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] FFPE cell pellet prep
Here's how I do it. I hate the lens paper method. I transfer suspended cells from a T75 flask to a 50 ml Falcon tube and centrifuge at 400 X g for 5 minutes. Aspirate most of the media so that cells plus media are about 1 ml. Transfer cells plus media to a 1.7 ml microcentrifuge tube. Pellet cells at 400 x G for 5 minutes. Rinse with PBS if needed. Suspend cells in NBF and fix over night. Next day, pellet cells and resuspend them in a small amount (100-200 microliters) of Histogel or Agar. When the gel cools cut the microcentrifuge tube open with a scalpel or razor blade. Now you have a nice cell pellet shaped like the bottom of your centrifuge tube, and you can treat it just like any piece of tissue. Pop it in a tissue processing cassette and then to your ethanol dehydrating steps. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Wed, 12 May 2010 06:50:14 -0700 From: kmerriam2...@yahoo.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] FFPE cell pellet prep Sorry - I forgot to put a subject line. Good morning, I am trying to make some nice FFPE cell pellet blocks, but I seem to lose a lot of cells along the way (especially when trying to take the cells out of the tube). We are fixing the cells in NBF, spinning them down, adding 70% and spinning down again. At that point, I am trying to scoop out the cells (with a weighing scoop) and wrap them in lens paper for processing. I am losing a lot of the cells during this step, because the pellet is not quite solid. Do you think it would be OK to let the cells air-dry a bit and then take them out for processing? I know this goes against everything I was taught in histology, but I am really at a loss here. Anyone have any hot tips for me? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ The New Busy is not the old 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_3___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Antigen retrieval question
Perhaps the antigen is soluble and diffused out of the tissue into the buffer? Jerry Ricks Research Scientist University of Washington Department of Pathology histonet@lists.utsouthwestern.edu Perhaps reversal is not the right word...slides were left overnight in buffer w/ tween. We got very minimal faint staining compared to previous runs done in one day. Brett -Original Message- From: Morken, Tim [mailto:timothy.mor...@ucsfmedctr.org] Sent: Tuesday, October 20, 2009 2:29 PM To: Connolly, Brett M; histonet@lists.utsouthwestern.edu Subject: RE: Antigen retrieval question Brett, how long? I've left them overnight before proceeding without any problems. It would not be re-fixation as with formalin. However, if it is in a detergent buffer it might cause some other kind of denaturation. Tim Morken Supervisor, Histology / IPOX UCSF Medical Center San Francisco, CA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M Sent: Tuesday, October 20, 2009 11:22 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Antigen retrieval question Has anyone experienced a reversal of citrate HEIR unmasking do to a prolonged delay in continuing the experiment after the retrieval step? Brett M. Connolly, Ph.D. Research Fellow, Imaging Dept. Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com _ Hotmail: Trusted email with powerful SPAM protection. http://clk.atdmt.com/GBL/go/177141665/direct/01/___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Antibody labeling
I have only done conjugation of biotin, HRP, and Alk-Phos. But I have discovered a new kit that makes the labeling process a snap. It is the Lynx kit from Serotec. They also have fluorophore labeling kits. It is fast, easy and very efficient. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Tue, 22 Sep 2009 12:32:20 -0700 From: jengirl1...@yahoo.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Antibody labeling Hello fellow Histonetters! I was wondering if any of you have ever labeled an antibody with fluorescence. If so which kit and protocol did you use? Or did you send the antibody out to be labeled? I'm hoping that you guys can help me out yet once again! Thank you all so much in advance. I really appreciate all of your help. Jennifer Sipes Johns Hopkins University Baltimore, MD __ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Microsoft brings you a new way to search the web. Try Bing™ now http://www.bing.com?form=MFEHPGpubl=WLHMTAGcrea=TEXT_MFEHPG_Core_tagline_try bing_1x1___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Black Silicone Rubber forfilling Dissecting Dishes?
I have some glass petri dishes with a black silicone rubber filling that work great for pinning and dissecting small animal tissues. The rubber works much better than the wax. Problem is, I don't know where the stuff came from, so I can't make any more dissecting plates. Does anyone know where I might buy the liquid for pouring the plates with? Thanks, Jerry Ricks Research Scientist University of Washington Department of Pathology _ With Windows Live, you can organize, edit, and share your photos. http://www.windowslive.com/Desktop/PhotoGallery___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Celloidin Sections.
Hi Barry, I am studying the development of atherosclerotic lesions in mouse brachiocephalic artery--I typically use 5 micron sections. Paraffin sections have given me mostly good, frequently excellent and occasionally spectacular results, morphology-wise. Oh, and occasionally poor results, too. I would prefer to increase the ration of spectacular/merely OK results. I read an article where the authors describe getting better fine morphology with celloidin than paraffin. Atheromas, like cochlea are delicate structures with fine detail, and they can easily become detached from the artery wall. After reading the article, I can't help but wonder if a different embedding media might give me even better results than I am already getting. Would I need a special microtome for celloidin? One reason I don't want to go with methacrylate is I don't have budget for new equipment right now. Jerry Ricks Research Scientist University of Washington Department of Pathology Effects of Fixative and Embedding Medium on Morphology and Immunostaining of the Cochlea Audiol Neurootol. 2009 ; 14(2): 78–87 From: barry.r.ritt...@uth.tmc.edu To: histonet@lists.utsouthwestern.edu Date: Mon, 20 Jul 2009 19:14:58 -0500 Subject: RE: [Histonet] Celloidin Sections. Jerry depends on whether you are talking about celloidin or Low Viscosity Nitrocelulose. There are techniques in the literature to cut celloidin sections as thin as 4 microns. For LVN you need much thicker sections as this plastic is much less sturdy and has a tendency to fragment if too thin. The questions I ask is why you need celloidin sections and what tissue are you thinking about for this technique? Barry From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of JR R [rosenfeld...@hotmail.com] Sent: Monday, July 20, 2009 6:42 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Celloidin Sections. I am curious--How thin can celloidin sections be cut? I've heard that 30 microns is a standard thickness. No way to make 5 micron sections? Thanks, Jerry Ricks Research Scientist University of Washington Department of Pathology _ Windows Live™ SkyDrive™: Store, access, and share your photos. See how. http://windowslive.com/Online/SkyDrive?ocid=TXT_TAGLM_WL_CS_SD_photos_072009___ 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 _ Bing™ brings you maps, menus, and reviews organized in one place. Try it now. http://www.bing.com/search?q=restaurantsform=MLOGENpubl=WLHMTAGcrea=TXT_MLOGEN_Local_Local_Restaurants_1x1___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] HELP Starting a lab
Pretty good list. Don't forget first aid kits and spill kits. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Mon, 20 Jul 2009 14:26:57 -0400 From: ktut...@umm.edu To: histonet@lists.utsouthwestern.edu Subject: [Histonet] HELP Starting a lab I have a friend who is a Pathologist and starting a new lab. She sent me a list of start up supplies and equipment that she needs to purchase. Can I get some input on whats missing from her list, I've added a few things but Im going blank. Its a hisology lab for a urology practice if that helps. Thank you in advance! PLEASE VENDORS DONT CALL OR EMAL ME Equipment: Negative pressure hood for grossing of specimens (grossing station) Refrigerator w/freezer or separate freezer Cassette labeler Tissue processor Embedding Stations Microtomes Waterbaths, 1 per microtome Automatic HE stainer A scale and a mixing/heating plate Manual staining dishes, and slide holders that fit in them. Slide and block storage system Supplies: Safety equipment like goggles and sleeve covers, aprons, a flame cabinet for flammable liquids acid cabinet? Blades Forceps Rulers Hemostats Scissors Glassware like flasks, beakers, graduated cylinders, and a funnel.Pipettes and tips. Reagents: Formalin 70% etoh 95% etoh 100% etoh xylene paraffin Hematoxylin stain Eosin stain Kimberly C. Tuttle HT (ASCP) Pathology Biorepository and Research Core University of Maryland Room NBW58, UMMC 22 S. Greene St Baltimore, MD 21201 (410) 328-5524 (410) 328-5508 fax Please consider the environment before printing this e-mail. This e-mail and any accompanying attachments may be privileged, confidential, contain protected health information about an identified patient or be otherwise protected from disclosure. State and federal law protect the confidentiality of this information. If the reader of this message is not the intended recipient; you are prohibited from using, disclosing, reproducing or distributing this information; you should immediately notify the sender by telephone or e-mail and delete this e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Windows Live™ Hotmail®: Search, add, and share the web’s latest sports videos. Check it out. http://www.windowslive.com/Online/Hotmail/Campaign/QuickAdd?ocid=TXT_TAGLM_WL_QA_HM_sports_videos_072009cat=sports___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Celloidin Sections.
I am curious--How thin can celloidin sections be cut? I've heard that 30 microns is a standard thickness. No way to make 5 micron sections? Thanks, Jerry Ricks Research Scientist University of Washington Department of Pathology _ Windows Live™ SkyDrive™: Store, access, and share your photos. See how. http://windowslive.com/Online/SkyDrive?ocid=TXT_TAGLM_WL_CS_SD_photos_072009___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Promega TUNEL assay...Or any TUNEL Assay
I don't like TUNEL on FFPE tissue. Way too many false positives. And yes, I've tried titrating down the concentration of TdT and substrate. Then I found out that formaldehyde causes DNA strand breaks. In other words for your positive control, you can used formalin fixed tissue, because formalin has the same effect as DNAase. The other thing problem with TUNEL on FFPE tissue is that the TUNEL assay detects DNA strand breaks. Any tissue, fixed of frozen, that is on a slide HAS been subjected to another procedure that causes double tranded DNA breaks--a procedure called microtomy. When a microtome bvlade passes through the nucleus of a cell it breaks a lot of DNA strands. And every one of them is detectable by TUNEL. I've heard of people getting rerasonable results with whole cells and frozen tissues, by for FFPE tissue, my current philosophy is: It is an assay that CANNOT work, even in principle. 'Course, I've been wrong about other things, I'm open to persuasion. Jerry Ricks Research Scientist University of Washington Department of Pathology _ Lauren found her dream laptop. Find the PC that’s right for you. http://www.microsoft.com/windows/choosepc/?ocid=ftp_val_wl_290___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Good Perfusion...
No, I wouldn't nick the atrium. Instead, cut the femoral artery at the groin. And of course, you cannot perfuse a living, anesthetized animal. That would be unethical and also illegal. Jerry Ricks Research Scientist University of Washington Department of Pathology _ Insert movie times and more without leaving Hotmail®. http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_Tutorial_QuickAdd_062009___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Good Perfusion--I stand corrected.
I stand correct on the perfusion under anesthesia issue. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Tue, 30 Jun 2009 12:37:20 -0700 Subject: Re: [Histonet] Good Perfusion... From: chana.de.w...@gmail.com To: jf...@gladstone.ucsf.edu CC: rosenfeld...@hotmail.com; histonet@lists.utsouthwestern.edu Histonetters, Perfusion under deep anesthesia is most certainly not unethical NOR illegal, and, in fact (as mentioned by Jo Dee), it is necessary for optimal perfusion and fixation -- the intracellular ischemic cascade begins immediately upon circulatory arrest, setting off a chain of events highly detrimental to subsequent perfusion. Indeed, ischemia quickly leads to the no-reflow phenomenon, effectively guaranteeing that you are not perfusing all tissues adequately at all! It is therefore *most* beneficial to begin perfusion with a beating heart, and every perfusion protocol I have ever worked under requires it (especially if the tissues are to be used for EM). I perform around 5-10 perfusions per week *specifically* to study the effects of ischemia on reperfusion and neural ultrastructure. Of course, you should check with your institution's particular rules and regulations, but perfusion begun under anesthesia is scientifically justified for the reasons mentioned above and should therefore be relatively easy to have approved by your IACUC. Sincerely, Chana de Wolf On Tue, Jun 30, 2009 at 11:37 AM, Jo Dee Fish jf...@gladstone.ucsf.edu wrote: Dear Jerry and histonetters, I don't believe this is unethical or illegal. It is written into our IACUC approved protocols as follows: Chemical Method of Euthanasia: Perfusion under general anesthesia, Avertin or Halothane induced. Bilateral thoracotomy. Could it only be forbidden by your facility? I wonder if other facilities have rules against such things. But we insist that our investigators use perfusion under anesthsia because the deeper tissues are much better fixed and blood removal is more thorough and complete by this method. Jo Dee ~~Jo Dee Fish~~ Senior Research Technologist The J. David Gladstone Institutes Co-manager Histology and Microscopy Core Telephone: (415) 734-2567 Fax: (415) 355-0824 E-mail: jf...@gladstone.ucsf.edu Mailing address: The J. David Gladstone Institutes 1650 Owens Street San Francisco, CA 94158 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of JR R Sent: Tuesday, June 30, 2009 10:49 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Good Perfusion... No, I wouldn't nick the atrium. Instead, cut the femoral artery at the groin. And of course, you cannot perfuse a living, anesthetized animal. That would be unethical and also illegal. Jerry Ricks Research Scientist University of Washington Department of Pathology _ Insert movie times and more without leaving HotmailR. http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_Tutori al_QuickAdd_062009___ 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 _ Insert movie times and more without leaving Hotmail®. http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_Tutorial_QuickAdd_062009___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] processing v-e-r-y tiny samples
Well, you could always hand process them in test tubes. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Thu, 19 Mar 2009 16:39:14 -0400 From: bryan.wat...@parkview.com To: histonet@lists.utsouthwestern.edu; algra...@u.arizona.edu Subject: Re: [Histonet] processing v-e-r-y tiny samples CC: I may never complain about tiny GI or bronch biopsies ever again! Andrea Grantham algra...@u.arizona.edu 3/19/2009 11:51 AM Good Morning! In keeping with the weirdness of the projects I get in this lab today my question is about processing mosquito GI tracts. I have a processing schedule - that is not the problem. I'm wondering if anybody out in histoland has a suggestion for what kind of cassette to use. I was thinking of the histoscreen cassette because these GI tracts are so thin (I think thinner than a hair)and I don't want to wrap them or use sponges because I'm afraid that I'll loose them or crush them. Any ideas? Andi . : Andrea Grantham, HT(ASCP) Dept. of Cell Biology Anatomy : : Sr. Research Specialist University of Arizona : : (office: AHSC 4212) P.O. Box 245044 : : (voice: 520-626-4415)Tucson, AZ 85724-5044USA : : (FAX: 520-626-2097) (email: algra...@u.arizona.edu) : :...: http://www.cba.arizona.edu/histology-lab.html ___ 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 _ Internet Explorer 8 – Now Available. Faster, safer, easier. http://clk.atdmt.com/MRT/go/141323790/direct/01/___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] RE: testing cutting ability--This is starting to Really Annoy me.
Instead of being so offended, why you just try to find out how different is academia from an AP histology lab? René J. I believe that I have already described an understanding that research is different than clinical. I did actually start off as a nurse way back when. Human dignity is the same everywhere though. It sounds like clinical labs mostly treat their Histotechs like dogs--starting with the interview, and continuing throughout the Tech's career. And I find that offensive. Jerry Ricks Research Scientist University of Washington Department of Pathology --- On Mon, 2/23/09, JR R rosenfeld...@hotmail.com wrote: From: JR R rosenfeld...@hotmail.com Subject: [Histonet] RE: testing cutting ability--This is starting to Really Annoy me. To: histonet@lists.utsouthwestern.edu Date: Monday, February 23, 2009, 2:58 PM All this talk of having people actually section as part of the interview process is offensive to me. I have hired and trained lots of histotechnologists. Mostly I train them myself, from scratch, but sometimes I hire them pre-trained. Then of course, I re-train them anyway so they can do the work to the specs of my lab. Any deficiencies they have will be corrected through training. By me. At my University there is a formal 30 day (maybe it's longer, I can't recall) probationary period for new hires. That's good enough. Why in the world would I need to have them section as part of an interview process? I am also offended by the blocks per hour language. Partly it's because in my lab we do exhaustive serial sectioning, so I consider a tech who gives me one block every half-hour to be doing a good job. Maybe it's different in clinical work, but...20 blocks per hour? And you expect quality work? Jeez, are clinical histopath labs just sweat-shops? It's no wonder histopathologists feel overworked and underappreciated. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Mon, 23 Feb 2009 10:14:06 -0500 From: trathbo...@somerset-healthcare.com To: rjbu...@yahoo.com; histonet@lists.utsouthwestern.edu; tbr...@holyredeemer.com Subject: RE: [Histonet] RE: testing cutting ability during an interview CC: We also have the staff talk to the applicant during this process. It gives you a good idea if the person can talk and cut at the same time. The last thing anyone wants is a histotech that has to stop sectioning during a conversation! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]on Behalf Of Rene J Buesa Sent: Monday, February 23, 2009 10:05 AM To: histonet@lists.utsouthwestern.edu; Terri Braud Subject: Re: [Histonet] RE: testing cutting ability during an interview I for one always required any applicant to prepare 20 slides stained with HE. I can assure that I selected difficult blocks to cut and the applicant was required to sign a disclaimer that included that s/he he knew how to section and avoid injuries. The whole process was timed (to get a first idea about productivity) and I evaluated and graded the slides at the end. The results were used as one of the elements to decide about offering the position (the fundamental) but I waited until all the applicants had completed the tests so sometimes the applicant had to be contacted a few days later to let him/her know about the results. René J. --- On Mon, 2/23/09, Terri Braud tbr...@holyredeemer.com wrote: From: Terri Braud tbr...@holyredeemer.com Subject: [Histonet] RE: testing cutting ability during an interview To: histonet@lists.utsouthwestern.edu Date: Monday, February 23, 2009, 8:57 AM From a recent digest: If you want to know if someone you are interviewing can really section or stain, set them down at a microtome during the interview process, and watch them. I have a question about the following statement plucked from a recent digest. What are the legal ramifications if a person cuts themselves during an interview? We've had this discussion at my place of employment and came to the decision that it would leave us open to a legal liability. I would love to hear some discussion on this subject, as well as any experiences that others have had. Terri Terri L. Braud, HT(ASCP) Anatomic Pathology Supervisor Laboratory Holy Redeemer Hospital and Medical Center - CONFIDENTIALITY NOTICE: This E-Mail is intended only for the use of the individual or entity to which it was sent. It may contain information that is privileged and/or confidential, and the use or disclosure of such information may also be restricted under applicable federal and state law. If you received this communication in error, please do not distribute
RE: [Histonet] sucrose
If you work in a clinical lab, then yes, there are reasons to use reagent or better grade sugar. Yes, as in Heck yes. Composition, Consistency, Legality, and Ethics. A scientist wants the experiment to have controlled conditions. That's especially true for you clinical science types. You want the test to give the same results every time. Reagent grade materials help ensure that. Next, in a clinical lab, it's probably required. Reagents used for clinical testing need to be traceable and of known composition. Next, people's lives actually depend on the results that come back from clinical labs. So you don't want to screw around with questionable materials. That would be wrong. Finally, Sigma sells reagent grade sucrose for $60.00 per kilogram. That's pretty darn cheap. What clinical lab can't afford a lousy 60 bucks for a kilogram of reagents?! If you work in a Research Lab, like I do, then feel free to go ahead and take your chances. But even we lowly research scientists like to get consistent results. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Mon, 23 Feb 2009 10:52:29 -0700 From: ejsch...@ucalgary.ca To: histonet@lists.utsouthwestern.edu Subject: [Histonet] sucrose hi, is there any reason that using ordinary table sugar is less desirable than some formulation of Mol Bio grade sucrose when preparing a specimen for cryrosectioning? Eric ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Windows Live™ Hotmail®…more than just e-mail. http://windowslive.com/howitworks?ocid=TXT_TAGLM_WL_t2_hm_justgotbetter_howitworks_022009___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: testing cutting ability--This is starting to Really Annoy me.
All this talk of having people actually section as part of the interview process is offensive to me. I have hired and trained lots of histotechnologists. Mostly I train them myself, from scratch, but sometimes I hire them pre-trained. Then of course, I re-train them anyway so they can do the work to the specs of my lab. Any deficiencies they have will be corrected through training. By me. At my University there is a formal 30 day (maybe it's longer, I can't recall) probationary period for new hires. That's good enough. Why in the world would I need to have them section as part of an interview process? I am also offended by the blocks per hour language. Partly it's because in my lab we do exhaustive serial sectioning, so I consider a tech who gives me one block every half-hour to be doing a good job. Maybe it's different in clinical work, but...20 blocks per hour? And you expect quality work? Jeez, are clinical histopath labs just sweat-shops? It's no wonder histopathologists feel overworked and underappreciated. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Mon, 23 Feb 2009 10:14:06 -0500 From: trathbo...@somerset-healthcare.com To: rjbu...@yahoo.com; histonet@lists.utsouthwestern.edu; tbr...@holyredeemer.com Subject: RE: [Histonet] RE: testing cutting ability during an interview CC: We also have the staff talk to the applicant during this process. It gives you a good idea if the person can talk and cut at the same time. The last thing anyone wants is a histotech that has to stop sectioning during a conversation! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]on Behalf Of Rene J Buesa Sent: Monday, February 23, 2009 10:05 AM To: histonet@lists.utsouthwestern.edu; Terri Braud Subject: Re: [Histonet] RE: testing cutting ability during an interview I for one always required any applicant to prepare 20 slides stained with HE. I can assure that I selected difficult blocks to cut and the applicant was required to sign a disclaimer that included that s/he he knew how to section and avoid injuries. The whole process was timed (to get a first idea about productivity) and I evaluated and graded the slides at the end. The results were used as one of the elements to decide about offering the position (the fundamental) but I waited until all the applicants had completed the tests so sometimes the applicant had to be contacted a few days later to let him/her know about the results. René J. --- On Mon, 2/23/09, Terri Braud tbr...@holyredeemer.com wrote: From: Terri Braud tbr...@holyredeemer.com Subject: [Histonet] RE: testing cutting ability during an interview To: histonet@lists.utsouthwestern.edu Date: Monday, February 23, 2009, 8:57 AM From a recent digest: If you want to know if someone you are interviewing can really section or stain, set them down at a microtome during the interview process, and watch them. I have a question about the following statement plucked from a recent digest. What are the legal ramifications if a person cuts themselves during an interview? We've had this discussion at my place of employment and came to the decision that it would leave us open to a legal liability. I would love to hear some discussion on this subject, as well as any experiences that others have had. Terri Terri L. Braud, HT(ASCP) Anatomic Pathology Supervisor Laboratory Holy Redeemer Hospital and Medical Center - CONFIDENTIALITY NOTICE: This E-Mail is intended only for the use of the individual or entity to which it was sent. It may contain information that is privileged and/or confidential, and the use or disclosure of such information may also be restricted under applicable federal and state law. If you received this communication in error, please do not distribute any part of it or retain any copies, and delete the original E-Mail. Please notify the sender of any error by E-Mail. Thank you for your cooperation. ___ 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 CONFIDENTIALITY NOTICE This message and any included attachments are from Somerset Medical Center and are intended only for the addressee. The information contained in this message is confidential and may contain privileged, confidential, proprietary and/or trade secret information entitled to protection and/or exemption from disclosure under applicable law. Unauthorized forwarding, printing, copying, distribution, or use
[Histonet] RE: equation problem PLEASE help
Hi Eva. Here’s how I would do it. (1 mole/1064 grams)*(.001 gram/ml)= 9.4 X 10^-7 mole/ml Concentration 1*Volume 1= Concentration 2 * Volume 2, or C1V1=C2V2 C1= 9.4 X 10^-7 mole/ml C2= 10X 10^-9 mole/ml V2= 2 ml Solving for V1... V1= (C2V2)/C1 V1= (10 X 10^-9 mole/ml*2.0 ml)/ 9.4 X 10^-7 mole/ml=.021 ml You need about 21 microliters of stock solution to make 2 mls of working solution. Use a calculator or Excel to get as many decimal places as you need. Jerry Ricks Research Scientist University of Washington Department of Pathology From: e...@georgetown.edu To: histonet@lists.utsouthwestern.edu Date: Wed, 7 Jan 2009 16:46:23 -0500 Subject: [Histonet] equation problem PLEASE help Good afternoon, I am hoping someone out there will take pity on a mathematically challenged individual such as myself. I have been trying for hours to wrap my head around this equation and am now getting to the point where I am more confused than ever. Please help me. The protocol calls for 10nmol of a substance per 1ml needed. It comes in a 1mg/ml solution and has a molecular weight of 1064g/mol. How do I do this? If for example I needed 2ml of the solution... The clearer the explanation the better. I really want to understand the calculation not just have an answer. PLEASE HELP ME. Thank you, Eva ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Windows LiveTM Hotmail®: Chat. Store. Share. Do more with mail. http://windowslive.com/explore?ocid=TXT_TAGLM_WL_t1_hm_justgotbetter_explore_012009___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Differences between WB antibody and IHC antibody
When I'm trying a new antibody for IHC, I will typically test several concentrations of the antibody and ALSO several different antigen retrieval methods side-by side. I typically do Heat Induced Epitope retrieval at pH 6.0 and 9.0, and also trypsin digestion for 30 minutes. Jerry Ricks Research Scientist University of Washington Department of Pathology Date: Fri, 2 Jan 2009 17:14:12 -0500 From: anh2...@med.cornell.edu To: talulahg...@gmail.com; histonet@lists.utsouthwestern.edu; ti...@foxmail.com Subject: Re: [Histonet] Differences between WB antibody and IHC antibody CC: Whether or not an antibody can be used for western vs IHC depends on many factors. Some antibodies can be used for both, some can be only used for one vs another, and some cannot be used for either. There are quite a few factors to consider and here are a few from the top of my head (and I am hoping people can add to this list): (1) Epitope recognition Western blotting often times (but not always of course) involves the denaturation of proteins as well as breaking of disulfide bonds etc. Samples are often boiled in denaturing agents such as beta-mercaptoethanol or DTT and are run in SDS containing buffers and gels. In doing this certain epitopes may be revealed enabling a specific antibody to work on western vs IHC. Along those same lines, this denaturation of proteins can also destroy epitopes, because proteins are no longer in their native conformations, disabling the efficacy of certain antibodies. Similarly, IHC involves fixation, processing, sectioning, heat retrieval etc. Fixation alone can alter epitopes dramatically thereby precventing a certain antibody from binding. On the contrary, proteins are often well preserved in a native conformation in IHC (especially in fresh frozen sections). (2) Access Because western blotting involves protein lysates of cells which have been busted open by a variety of methods, all of the proteins in a cell should be accessible to the antibody. In IHC on sections or on coverslips etc, the cell structures are largely preserved intact. Therefore certain cell compartments may need further processing to grant access of the big bulky antibody to the inside of the compartments. Hence part of the reason why we do antigen retrieval with enzymes and detergents etc. Similarly because in western, one might spin out fractions before running on a gel if you are searching for a nuclear protein or other protein localized to a certain compartment you must be sure you actually have the compartment preserved in a western prep where you always would have it present in an intact IHC section. (3) Sensitivity Western blotting is superior to run-of-the-mill IHC in detection sensitivity. This is even more obvious when one considers how easy it is to interpret weak or sparse IHC staining as background. Background is much easier to interpret in a western blot. Therefore an antibody which works beautifully in WB may work in IHC but the proteins might be too sparse or too few in number to be visualized. (4) Polyclonal vs. monoclonal If an antibody is a polyclonal antibody (pAb) it will likely have a better chance to work across a broad spectrum of applications. This is because a polyclonal antibody preparation is actually a combination of different Ig's recognizing more than one epitope (although it is important to know that there may be a predominant Ig present or the pAb could have been purified in a way to enrich for a particular antigen which may alter it's ability to work in a broad range of apps). That's all I can think of for now, and I hope this makes sense ... but as someone else said, it's all empirical and has to be tested in each person's lab. Happy New Year! If anyone replies with posting to the list, please forward it. I've always wondered the same thing. On Fri, Jan 2, 2009 at 8:15 AM, TF ti...@foxmail.com wrote: Hi Just wondering whether the antibody for WB (only WB, IP on datasheet) can be used for IHC use? If not, why? Can I just increase the concentration for IHC application? I want to learn more about the underlying production processes. Thanks! 2009-01-02 TF -- ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ It’s the same Hotmail®. If by “same” you mean up to 70% faster. http://windowslive.com/online/hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_broad1_122008___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
