[Histonet] RE: Autofluorescence problem on atheroma - TUNEL

[C.M. van der Loos]

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 <eberledelphine <@t> hotmail.com>
Subject: [Histonet] Autofluorescence problem on atheroma - TUNEL
staining
To: histonet histonet <@t> 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.eberle <@t> ucsfmedctr.org