[Histonet] Antibody for canine IgG
I'm looking for an antibody that works for canine IgG, to be used for FFPE tissue IHC, and which should react with both IgG1 and IgG2. Ideally mouse monoclonal. Does not have to be specific to dog, as long as it reacts with it adequately in IHC. Any recommendations ? _ Jean-Martin Lapointe AccelLAB Inc ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
re: [Histonet] clostridium and bacterial spores in tissue sections
Hi Liz, I'm not sure why a good old Gram (or Brown and Brenn) wouldn't work for your sections. I have visualized clostridia with such stains in the past, and the terminal spores were easily visible. __ Jean-Martin Lapointe, DMV, MS, dACVP Vice-President, Pathologie AccelLAB Inc 1635 Lionel-Bertrand, Boisbriand Québec, Canada J7H 1N8 tel: 450-435-9482 ext.247 fax: 450-435-4795 jm.lapoi...@accellab.com --- On Sat, 1/14/12, Elizabeth Chlipala wrote: From: Elizabeth Chlipala Subject: [Histonet] clostridium and bacterial spores in tissue sections To: "histonet@lists.utsouthwestern.edu" Date: Saturday, January 14, 2012, 12:58 PM Hello everyone I need a bit of help. I have to stain for clostridium and bacterial spores in tissue sections. I have come across a few references to staining these on smears but not on tissue sections. I did find one reference that used Ziehl-Neelsen to stain terminal spores from clostridium tetani. Does anyone out there know if the stain they use in microbiology for smears will work on tissue sections? Any advice would be appreciated. Thanks in advance Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308-1592 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell www.premierlab.com<http://www.premierlab.com> ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. & neg. control question
I can certainly agree with that. Whether it’s the patient’s sample or the known positive control that are stained without primary, either would fulfill the purpose of detecting non-specific positive staining. Jean-Martin From: Angela Bitting [mailto:akbitt...@geisinger.edu] Sent: Thursday, May 19, 2011 4:50 PM To: Jean-Martin Lapointe; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. & neg. control question I agree with the majority. A patient slide stained without primary antibody, and a patient section with primary antibody and a known positive control tissue, on the same slide when possible, is sufficient. I do agree, however, that the use of a negative tissue control is important in your initial antibody optimization and validation. Angela Bitting, HT(ASCP), QIHC Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 email. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC pos. & neg. control question
I think that this is indeed what is happening here, that there is confusion between a negative control stain (positive tissue, stained without the primary ab) and a negative control tissue (tissue known to not express the marker, stained normally). I had assumed we were talking about the former, because this is what the pathologist cited by Curt wrote in his email. While a known negative control tissue is useful in an IHC run, I am somewhat alarmed to gather from the responses sent that a section without primary is NOT being included in standard IHC runs at your respective labs. To me, this is an absolute necessity in order to properly evaluate IHC results. But that might be because I work in research rather than in a clinical setting, where i'm assuming that priorities are different. Also, I do not agree with Glen's post, for the reasons outlined by Pete. Jean-Martin -- Message: 11 Date: Thu, 19 May 2011 13:37:38 -0500 From: "Sebree Linda A" Subject: RE: [Histonet] IHC pos. & neg. control question To: "Dawson, Glen" , Message-ID: Content-Type: text/plain; charset="us-ascii" I basically agree with you Glen. I think some people are mixing up Negative Reagent Controls (substituting negative serum, Ab diluent, etc. for antibody) and Negative Tissue Controls (substituting a tissue known to be negative for the antibody being run). It CAN be confusing. Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 1:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 -- Message: 12 Date: Thu, 19 May 2011 14:53:57 -0400 From: Mary Helie Subject: [Histonet] neg control To: histonet@lists.utsouthwestern.edu Message-ID: <4dd56745.3030...@yale.edu> Content-Type: text/plain; charset=ISO-8859-1; format=flowed What??? News to me as well. -- Message: 13 Date: Thu, 19 May 2011 14:01:45 -0500 From: Subject: [Histonet] Slides for IHC To: Message-ID: Content-Type: text/plain; charset="us-ascii" So I just cu
[Histonet] IHC pos. & neg. control question
Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin __ Jean-Martin Lapointe, DMV, MS, dACVP Vice-President, Pathologie AccelLAB Inc 1635 Lionel-Bertrand, Boisbriand Québec, Canada J7H 1N8 tel: 450-435-9482 ext.247 fax: 450-435-4795 jm.lapoi...@accellab.com -- Message: 24 Date: Thu, 19 May 2011 09:04:24 -0700 From: "Curt Tague" Subject: [Histonet] IHC pos. & neg. control question To: Message-ID: <019801cc163e$6c1487d0$443d9770$@ta...@pathologyarts.com> Content-Type: text/plain; charset="us-ascii" I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Tumors tumors everywhere
Hi Sarah, in a previous job my lab did extensive IHC staining on mouse xenografts of human tumors, like your tissue. We never used mouse monoclonal primaries on this, because of the background issue. Even with a kit designed to supposedly block the non-specific mouse cross-reactivity, the results were not good the few times we tried it. When using a rabbit poly (or rat mono) as primary, and the appropriate detection system, non-specific background was never a problem. So like Ashley says, your problem is probably with your MACH3 detection system, which seems to pick up mouse antigens. Use a rabbit-specific one, and your problem should go away. Good luck, __ Jean-Martin Lapointe, DMV, MS, dACVP Vice-President, Pathologie AccelLAB Inc 1635 Lionel-Bertrand, Boisbriand Québec, Canada J7H 1N8 tel: 450-435-9482 ext.247 fax: 450-435-4795 jm.lapoi...@accellab.com -- Message: 10 Date: Mon, 2 May 2011 11:46:29 -0500 From: "Troutman, Kenneth A" Subject: Re: [Histonet] Tumors tumors everywhere To: "Histonet@lists.utsouthwestern.edu" Message-ID: <7b310892042da74cb3590053f424cfe613d63cd...@its-hcwnem06.ds.vanderbilt.edu> Content-Type: text/plain; charset="us-ascii" Hi Sarah, Biocare's MACH3 is designed to work with mouse or rabbit antibodies, so it is likely picking up some mouse antigens: http://biocare.net/products/detection/mach-3/ MACH 3(tm) MACH 3 is a two-step, biotin-free detection system which provides excellent specificity, sensitivity and nuclear staining for mouse or rabbit primary antibodies. I would choose a reagent that recognizes ONLY your rabbit antibody. Check out Biocare's Starr Trek components (like the Trekkie Rabbit link. I know the name is goofy... :)). They should be able to help you. Good luck. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 7 Date: Thu, 28 Apr 2011 15:16:16 -0500 From: Subject: [Histonet] Tumors tumors everywhere To: Message-ID: Content-Type: text/plain; charset="us-ascii" So I am staining tumors that were implanted as cells subdermally into mice. The cells are human. I am trying to do Caspase staining on these tumors. The primary is an anti-human rabbit polyclonal, and I am using a polymer (Biocare Mach3) in lieu of the secondary antibody. The background is through the roof!! Could the reason be that the tumor was grown in a mouse and is having cross reactivity somehow? What species antibody should I be using instead? All my mouse monoclonal antibodies work perfect on the tissue, it's this stupid rabbit polyclonal!! I am blocking endogenous enzymes (peroxidase etc., DAKO), avidin and biotin (just to see if that would help...it didn't), and protein block (it's literally an hour worth of blocking!!), developing with DAB (Dako) and hematoxylin counterstain. I am so confused as how to get this to work! Also, it isn't just this particular antibody it is any rabbit polyclonal I have tried. Could it be the polymer? It is the one that Biocare suggested? HELP!! Thanks in advance =) Sarah Goebel, 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] RE: Histonet Digest, Vol 87, Issue 5
Hi Shu, I would stay away from zinc fixatives if I were you. It does a very poor job of preserving tissue integrity. There's nothing too esoteric in the cells you need to stain, so formalin fixation should be fine for this. Jean-Martin Lapointe Accellab Inc -- Message: 2 Date: Wed, 2 Feb 2011 13:28:47 -0500 From: "Chen, Shu-Cheng" Subject: [Histonet] Rat lung histology for mast cells and eosinophils To: "histonet@lists.utsouthwestern.edu" Message-ID: <5d62649615faa6478f801a08d10e51855983c4a...@usctmxp51003.merck.com> Content-Type: text/plain; charset="us-ascii" Hi, We are called to support an asthmatic rat lung project and need to stain for mast cells, eosinophils, neutrophils and possibly other inflammatory cells, such as Th2 cells etc. Any suggestions you can give us in terms of fixatives and special stains or IHC are very much appreciated. From my search, it seems that Carnoy's fixative with toluidine blue is the way to go for mast cells. But can one clearly differentiate eos from neuts with this fixative or a formalin fixed H&E stained lung section? If we need to do Trichrome, PAS, May Grunwald Giemsa and H&E etc. what would be the best fixative to accommodate these stains? BTW, I heard that formalin free Zn-fixative is good for IHC. Will it be good for all these stains above? Sorry for so many questions. It shows how little experience I have in this area. Thank you, Shu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re: fixation question for IHC (Jan Berry)
I think it is a very small minority of markers that would be sensitive to the methanol residues present in formalin. So it depends on what you plan on staining. For general purposes, I would always go with formalin, unless there is a proven need for formaldehyde. __ Jean-Martin Lapointe, DMV, MS, dACVP Vice-President, Pathologie AccelLAB Inc 1635 Lionel-Bertrand, Boisbriand Québec, Canada J7H 1N8 tel: 450-435-9482 ext.247 fax: 450-435-4795 jm.lapoi...@accellab.com -- Message: 3 Date: Fri, 28 Jan 2011 13:15:19 -0500 From: Jan Berry Subject: [Histonet] fixation question for IHC To: histonet@lists.utsouthwestern.edu Message-ID: <1ba205bb-22b9-4167-a36d-01b7f3f04...@umich.edu> Content-Type: text/plain; charset=us-ascii Is there a difference between using paraformaldehyde and neutral buffered formalin when choosing a fixative for IHC? I would prefer to use formalin because of easier preparation, but am willing to put in the extra time to make fresh paraformaldehyde solution if there is a compelling reason. Jan Berry University of Michigan ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Immunohistochemistry in plastic sections
Hi Jan IHC on plastic-embedded specimens is difficult, I think the antigens can get rather damaged from the embedding and/or deplastifying processes. In our experience, it's possible to do some markers, while others are irretrievable. One thing I notice is that our protocol for deplastification calls for 2x MEA 20 minutes, while yours does 3x. Perhaps cutting down to 2x would be sufficient (your sections are thinner than ours, too), and would potentially cause less damage. Good luck __ Jean-Martin Lapointe, DMV, MS, dACVP AccelLAB Inc -- Message: 4 Date: Fri, 21 Jan 2011 09:41:39 -0500 From: Jan Berry Subject: [Histonet] Immunohistochemistry in plastic sections To: histonet@lists.utsouthwestern.edu Message-ID: Content-Type: text/plain; charset=us-ascii I'm wondering if anyone has experience with immunohistochemistry using plastic sections. I am currently working with 4um sections, and have tried doing staining with and without Dako antigen retrieval, but I am not getting very good results. Tissues (mostly mouse bones, but some soft tissues also) were fixed in PFA, cold embedded, and sections were deplastified using 3X 20 minutes methoxyethyl acetate and 2X 5minutes acetone, followed by 5 minutes in running dI water before beginning. I would appreciate any advice! Jan Berry, University of Michigan ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Phospho antibodies and fixation
Hi Ashley, I did a little bit of work on this a few years back. My belief is that the main difficulty with IHC for phospho-epitopes is not formalin fixation per se, but rather the instability of the phosphorylation. I used to work with rodent tumor models, where we collected solid tumors (or other tissues) just after sacrifice, and fixed them by formalin immersion. We found that the periphery of the tissues fixed this way expressed various phospho-epitopes quite well, but not the center. My interpretation was that by the time formalin had penetrated to the deep regions of the tissue, the phosphorylation had gone away (as you know formalin penetrates solid tissues fairly slowly). We proceeded to test matched tissue samples, fixed either in formalin at room temperature vs. cold formalin (formalin was at 4C at immersion, and the tissue was put in the fridge after sampling). We found that the cold formalin samples expressed phosphorylation sites fairly evenly, compared to only peripheral with the room temp samples. Presumably the cold temperature preserves the phosphorylation sites long enough to leave time for formalin to penetrate throughout. Consequently we integrated cold formalin fixation to all our IHC protocols for phospho-epitopes. Hope this helps, Jean-Martin Lapointe Accellab -Original Message- Good day colleagues, Does anyone have any information on phospho antibodies and fixation? Is there any reason to NOT fix specimens in formalin vs say, 70% EtOH? (and then process them through formalin later?) I can't seem to find any information on whether or not fixation in formalin does something strange to a phosphorylated protien and makes it a less accessible antigen. Also, on that same note, does retreival do anything to it? I am assuming that these antibodies go through the same testing for cross reactivity, etc (depending on the vendor) and are reliable when used properly (like any other antibody). I don't, however, know if there is enough of a difference in the epitope (the fact that it is phosphorylated) that would make it more susceptible to some strange cross linking with formalin (especiallly with phosphate buffered formalin). Any help with this topic would be greatly appreciated as I am uneducated in this area. Thanks, Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 615-343-9134 ___ 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 End of Histonet Digest, Vol 77, Issue 21 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Cd31 in pigs
Hi Joel, we have tried a few antibodies for CD31 in FFPE tissues in pigs and have had no luck. We currently use an antibody to vWF to stain pig endothelium. If someone has a working technique for CD31 in swine, I'd be happy to hear about it. Jean-Martin __ Jean-Martin Lapointe, DMV, MS, dACVP AccelLAB Inc Québec, Canada J7H 1N8 jm.lapoi...@accellab.com Date: Fri, 9 Apr 2010 09:40:00 -0400 From: "Joel Israel" Does anyone have a procedure that WORKS for cd31 in pigs? I can't seem to get it to work no matter what I do. Any help will be greatly appreciated. Thanks.- joel ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] frozen myocardium sections
Hi all, for a study we are freezing myocardium sections in OCT immersed directly in liquid nitrogen, without isopentane, because apparently isopentane quenches the fluorescence of the cells we need to detect in the tissue. Unfortunately the blocks tend to crack after freezing. Does anyone have a suggestion to avoid cracking ? thanks __ Jean-Martin Lapointe AccelLAB Inc jm.lapoi...@accellab.com <mailto:jm.lapoi...@accellab.com> ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] CD31 on rat brain
I would guess that there are problems with the antibodies you are using. SMA is usually a very robust antigen for IHC, if you're not getting any staining you're probably using the wrong antibody. You need an antibody whose specs indicate that it works on mouse, and that it works on fixed tissue. You also don't give details on your staining protocol - it could be a technical issue with that as well. To find proper antibodies, I have found the Biocompare website to be an excellent place to look. Jean-Martin Lapointe AccelLAB Inc -- Message: 11 Date: Sat, 7 Mar 2009 00:44:32 +0800 From: "TF" Subject: Re: RE: [Histonet] CD31 on rat brain [Histonet] staining brain vessels > > I was wondering if anybody might have an idea with the following > problem we are experiencing: we want to stain for blood vessels in > sections of mouse brain. Our experimental tissues have been fixed > overnight in 4% paraformaldehyde and have been sitting in PBS since. > We have tried staining with antibodies against desmin, SMA, and > collagen but we get NO specific signal. We recently tried a non-fixed > mouse brain and got desmin to work immediately. The problem is that > we need to use the fixed brains because they are our experimental > model and it would take too long (2 years to be exact) to generate the > same samples. If anybody has come across such a problem before, or > has a specific protocol for vessels that works on PFA fixed brain, we > would appreciate the suggestions! > thanks in advance! > Irini > - > Irini Skaliora, PhD > Investigator Cá¾½ (Assistant Professor) > Developmental Biology Division > Biomedical Research Foundation of the Academy of Athens (BRFAA) > Soranou Efessiou 4 > Athens 11527 > tel. +30-210-6597203 (office) > tel. +30-210-6597482 (lab) > fax. +30-210-6597545 > email: iskali...@bioacademy.gr ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: (no subject) (Geoff McAuliffe)
Tojo, Geoff provides very good pointers, but I think his option 3. is the correct one in your case, i.e. what looks like 'holes' are areas of glycogen and/or lipid that are unstained with the technique you are using. If your rats are not fasted prior to euthanasia, the cytoplasm of the hepatocytes will be filled with glycogen. Trying a PAS would likely confirm this, by staining the clear areas positive. Jean-Martin __ Jean-Martin Lapointe, DMV, dACVP AccelLAB Inc -- Date: Tue, 16 Sep 2008 14:17:48 -0400 From: Geoff McAuliffe <[EMAIL PROTECTED]> Subject: Re: [Histonet] (no subject) To: "TOJO (Torben Seested Johansen)" <[EMAIL PROTECTED]> Dear Tojo: Your problem could be due to 3 things. 1. Waiting too long to fix the tissue. 2. Freezing too slowly. 3. Interpreting the lack of staining of hepatocyte glycogen as an artifact. 1. You are waiting 5 minutes after death to fix the tissue. There is absolutely NO reason to spend 5 min pumping 100 ml of PBS through the circ. system. You will never wash out all of the blood and there is no need to. AND when you finally get to fixative, the flow of fixative is too low. Here is the solution to your problem. Use a 16 g needle in the L. vent. Pump 10-15 ml of room temperature PBS into the rat in 15-20 seconds. Pump 300 ml of room temperatue fix in 5 minutes. The fix is 4% paraformaldehyde for light microscopy. For transmission EM the fix is 2 or 3 or 4% paraformaldehyde with 1% glutaraldehyde. Buffer can be either phosphate or cacodylate. The advantage of cac. buffer is that you can add some calcium salts to stabilize membranes for EM (2milleMole) without percipitation.Yes, you can add Ca salts to phos. buffer but it will precipitate instantly (look up the solubility of CaPhosphate, or should I say the insolubility). Of course, cacodylate has arsenic in it so don't lick your fingers. Remove liver and cut into appropriate sized pieces. For light microscopy fix as long as possible, formalin reacts with tissue very slowly. Glutaraldehyde fixed much faster, an hour or two is plenty. Cryoprotect with sucrose. 2. Tissue must be frozen VERY QUICKLY with 2-methylbutane (isopentane) cooled with dry ice or liquid nitrogen otherwise you will have large holes due to ice crystal formation. 3. The stain you are using, Tol.Blue will not stain glycogen so you may have empty-looking areas because of this. Use Periodic acid Schiff for glycogen but NOT after glutaraldehyde fixation. Geoff TOJO (Torben Seested Johansen) wrote: > Hi, > I do not seem to be able to achieve acceptble morphology of perfused rat > liver. I seems as if some of the cytosol of the hepatocytes are "washed away" > as seen in toluidine blue stained cryo-sections (see image18.jpg). I am to > use the tissue cryo-sections in immunohistochemistry and transmission > electron microscopy. > > I my attempts to get an acceptable/good morphology I have tried fixation > buffers consisting of 2, 4, 6 or 8 % paraformaldehyde (w/wo 0.1% > glutaraldehyde). > > My fixing procedure is as follows (all at room temperature); > > a rat (~250g) is anasthestized and a perfusion needle is inserted into the > left ventricle. The atrium is cut and PBS is flushed thru the rat at 20ml/min > for 5min. The rat is thereafter fixated at 10ml/min for 10 min with fixation > buffer (I have so far tried 2, 4, 6 or 8 % paraformaldehyde (with and with > out 0.1% glutaraldehyde) in 0.1M cacodylatebuffer (pH 7.4) all with same > unacceptable result (see image18.jpg). The liver is cut into smaller pieces > (~2*2*2mm) post fixed for 1h in the respective fixative, transferred into > 2.3M sucrose for minimum 1h (preferably over night) and frozen in liquid > nitrogen. > > Any idea why teh cytosol of my hepatocytes seems to be "gone" (hydropic > degeneration?) ? > > I have searched the net and it seems as if theres a 1000's of different ways > of performing rat liver perfusion fixation. Some use cold buffers and others > also perfuse with sucrose solution (either in combination with the fixative > or with sucrose alone post-fixation) > > what can I do to try and optimise my perfusion ? > > __ > > Torben Seested Johansen > Post Doc > Exploratory ADME, Biopharmaceuticals ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
