Re: help with quenching autofluorescence
|From:||Karen Pawlowski <Karen.Pawlowski@worldnet.att.net>|
Some colleges of mine published an article on reducing auto-
fluorescence on paraformaldehyde fixed, frozen brain sections a while
ago. I think it might work for you too.
The article is Clancy, B. and Cauller, L. J., Reduction of background
auto fluorescence in brain sections following immersion in sodium
borohydride in Journal of Neuroscience Methods, 83(2):97-102, 1998.
They reference another article, Beisker W., Dolbeare F. and Gray J.W.,
An improved immunocytochemical procedure for high-sensitivity detection
of incorporated bromodeoxyuridine. Cytometry 8:235-239, 1987.
I haven't read this article, but it might prove the most useful to you.
In the first article, they floated their sections (50 micron sections of
cortex) in 0.1% sodium borohydride for 3 min. to 4 hrs. Be careful
how long you leave the sections in solution though, as the tissue might
start to look chewed up and come free from the slide.
I hope this helps you. I cna't comment on the similarity of the
staining patterns for Ki-67 and HP1 antigen in mouse tissue. Hopefully
someone else can help you with that.
Karen Pawlowski, PhD
"Zhao, Xinyu" wrote:
> Dear histonetters,
> I am working on a project using immunofluorescence staining to detect Ki-67 and HP1 antigen in mouse tissue. I got positive result but the autofluorescence background (in both red and green) is severe. And strangely, the staining patterns for those two antibodies are the same.
> My questions:
> 1. How can I quench the autofluorescence (the tissue was formalin-fixed and paraffin-embedded)?
> 2. Could my staining with Ki67 and HP1 be false positive?
> Any input will be great appreciated.
> Jasmine X.Y. Zhao
> The Wistar Insitute
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