Dear Delphine, Reading your message I was wondering in the first place why TUNEL? Cleaved caspase-3 antibody available from several vendors is doing a marvelous job and is way much easier than TUNEL. Besides that there is literature available that caspase-3 IHC demonstrates less false positive signal than TUNEL. Unfortunately, there are still old fashioned reviewers who stick to TUNEL as 'gold standard'. I guess that formalin-fixation of the cryo's is needed and I am almost sure that acetone is not working for TUNEL. Paraffin tissue sections are to my idea much better for TUNEL than cryo's. Having said that you better switch to enzymatic visualization rather than fluorescence. The formalin causes massive autofluorescence that is hard to kill. And if you get it killed you may ask yourself if your specific signal is perhaps also killed. Use tonsil (follicle center) as positive control for TUNEL or caspase-3. Hope this helps a bit. Cheers, Chris Chris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands
Date: Fri, 17 Sep 2010 21:13:21 +0200 From: delphine eberle <eberledelph...@hotmail.com> Subject: [Histonet] Autofluorescence problem on atheroma - TUNEL staining To: histonet histonet@lists.utsouthwestern.edu Hi, Autofluorescence in atheroma is a well known problem for immunofluorescence staining. I would love to hear input about how to reduce it to a minimum (if possible!). So far, we are trying to detect apoptotic cells in atheroma by TUNEL staining (Roche, TMRred) and we found the interpretation very difficult as some area of the tissue show red dots, tat could be considered as apoptotic bodies. We are working with frozen sections, fixing with Formalin, quenching with Glycine etc... -I was wondering if acetone fixation (or other fixation methods) could decrease atheroma autofluorescence by extracting lipids and others materials? -Also, I am not sure that acetone fixation is recommended for TUNEL staining? Did anybody try? -Are there around any approved ways to decrease autofluorescence in atheroma? (I can not find anything!) Thanks! Delphine Delphine Eberl� PhD UCSF Department of Vascular Surgery VA Medical Center - NCIRE Building 2 - room 410 4150 Clement Street San Francisco, CA 94121, USA Tel: 415 221 4810 ext.2984 Cell: 857 453 0821 delphine.ebe...@ucsfmedctr.org _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
