Patsy and Amos I can appreciate your comment but we have been using Dako's Envision+ Rabbit on mouse tissue for years now and have never seen any cross reactivity to mouse tissue, it's pretty much our go to detection system for rabbit antibodies (both monoclonal and polyclonal) on mouse tissue. We have however seen cross reactivity to porcine with Dako's envision +mouse and other companies anti-mouse polymer reagents. My suggestion if you have not already done this is to try to use some other retrieval methods such as pH6 or enzyme (proteinase K or pepsin or even no retrieval). Tissue fixation is important along with time an temp of retrieval - under fixation or over retrieval or a combination of both can cause problems. If we find we are getting some background we will run some more intense blocking - such as the following:
1. commercially available superblock - these can be competitive and may potentially cause decreased sensitivity so you will need to watch out on these 2. Normal serum - we normally use a commercial serum free protein block but on occasions we will use up to a 20% normal serum block for 30 to 60 minutes, if you want to continue to use the envision+ rabbit your choice would be 20% normal goat serum 3. Saturated Casein - we'll make this up in house All are worth a try, good luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -----Original Message----- From: ih...@googlegroups.com [mailto:ih...@googlegroups.com] On Behalf Of pru...@ihctech.net Sent: Tuesday, January 28, 2014 12:11 PM To: 'Amos Brooks'; histonet@lists.utsouthwestern.edu; 'ihcrg Group, (E-mail)' Subject: [IHCRG] RE: [Histonet] Mouse GranzymeB Amos u might have to get an anti rabbit link that has been mouse absorbed. Dako's anti rab envision is made in a goat but it is not mouse absorbed. Southern Biotec or Jackson Labs are really good places to get absorbed links. Still no reliable cd4 and cd8 for ffpe mouse tissue as far as I know, I have tried many that claim they work but have not consistently worked in my hands, we still do that with frozen mouse tissue not aldehyde fixed. Are u doing HIER with the EDTA ph9? In my experience especially with the higher ph u can over retrieve and cause undesirable nuclear staining. 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.com rueggihcconsultin...@outlook.com This email is confidential and intended solely for the use of the Person(s) ('the intended recipient') to whom it was addressed. Any views or opinions presented are solely those of the author. It may contain information that is privileged & confidential within the meaning of applicable law. Accordingly any dissemination, distribution, copying, or other use of this message, or any of its contents, by any person other than the intended recipient may constitute a breach of civil or criminal law and is strictly prohibited. If you are NOT the intended recipient please contact the sender and dispose of this e-mail as soon as possible. -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos Brooks Sent: Friday, January 24, 2014 10:09 AM To: histonet@lists.utsouthwestern.edu; ihcrg Group, (E-mail) Subject: [Histonet] Mouse GranzymeB Ok Musketeers, I am trying to detect cytotoxic T-Cells in formalin fixed paraffin embedded mouse liver. GranzymeB should do the trick. I have a rabbit anti mouse (and human and rat) GranzymeB from abCam (ab53097). The spleen that I ran as a control with it works fine. Nice T-cells and no signifigant background staining. The liver on the others hand has some lymphocyte staining, presumably cytotoxic T-cells as expected, but it looks like every hepatocyte nuclei are also picking it up. Grrr, right? If this antibody had been raised in mouse, I wouldn't be surprised. Also, if the spleen looked similar, I wouldn't be surprised. Do any of you have any ideas ab pi ut what this might be? Alternatively, does anyone have any ideas about another antibody that would work here? CD4 or CD8 would be great here, but its FFPE mouse, so sadly that's out (more grumbling). For the completionists out there, I used EDTA pH9, peroxide block and detected the antibody (diluted 1:800) with Envision (rabbit) and DAB from Dako. Any suggestions would be vastly appreciated, Happy Friday, Amos _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- -- You received this message because you are subscribed to the Google Groups "ihcrg" group. The IHC Resource Group is a standing committee within the National Society for Histotechnology. To post to this group, send email to ih...@googlegroups.com To unsubscribe from this group, send email to ihcrg+unsubscr...@googlegroups.com For more options, visit this group at http://groups.google.com/group/ihcrg?hl=en To contact the National Society for Histotechnology, email: hi...@nsh.org or call 443.535.4060. --- You received this message because you are subscribed to the Google Groups "ihcrg" group. To unsubscribe from this group and stop receiving emails from it, send an email to ihcrg+unsubscr...@googlegroups.com. For more options, visit https://groups.google.com/groups/opt_out. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
