Re: [Histonet] immunofluorescence problem

From:Geoff McAuliffe

Hi Stephanie:

Have you compared a NeuN to an adjacent section NeuN that was preceeded 
by HCl denaturation to see if the HCl really is the problem?
Can you use a fixative that does not require DNA denaturation or 
requires a milder treatement?
Can you do the NeuN, photograph the results, then do the other stains 
and photograph them? A final composite with all 3 antibodies can be made 
in PhotoShop.

Geoff wrote:

>I am doing triple labeling immuno fluorescence of mouse brain tissue and am having some difficulties.  My three markers are NeuN, GFAP, and BrdU.  Previously when I have triple labeled, I only get gfap and brdu fluorescence and no NeuN (or rarely and inconsistently).   I have found out that it is possible that the acid used to denature the DNA for BrdU may be interfering with NeuN antibodies.  I was wondering if anyone has also had this problem with NeuN.  Does anyone have any ideas to get around the NeuN/hydrochloric acid issue?
>Stephanie Lieblich
>Cellular Neurophysiology Lab
>University of British Columbia
>Vancouver, B.C., Canada

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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