immunofluorescence background

Andrew K. Groves grovesa at starbase1.caltech.edu
Sun Apr 2 18:40:56 EST 1995


> In article <3ln1sn$1q9 at news.duke.edu>,
> altschul at PROBLEM_WITH_INEWS_GATEWAY_FILE (Danny Altshuler) wrote:
> 
> > Hi, everybody
> > I'm doing some fluorescence using the biotynilated 
> > antibodies/streptavidin Texas Red. I'm using 4% parafolmadehyde and 0,5% 
> > Triton for the fixation /permeabilization steps.
> > My problem is in the background. I started using BSA 3% whitout luck and 
> > then switch to 5% calf serum. The background was reduced but is still there.
> > Please any suggestion or good protocols are wellcome
> > BTW these are NIH 3T3 cell lines
> 

It's not clear whether your problem is a non-specific binding of primary
antibody, or autofluorescence due to fixation. The former can be helped by
using species-specific normal serum, as another poster has pointed out.
Autofluorescence on tissue sections can be reduced by:

- fixing, processing, cutting and staining the tissue as quickly as
possible, as opposed to letting the tissue hang about for days.
- Including a small amount (0.02M) glycine in your buffers, which can help
to neutralise aldehyde fluorescence.

One additional point concerning streptavidin conjugated fluorophores.  It
is best *not* to apply these reagents in buffers containing either serum
or BSA. These both contain biotin, and will deplete the amount of free
streptavidin-fluorophore conjugate in solution, thus giving less intense
signal.

Hope this helps,

Andy

-- 
Andy Groves
Division of Biology, 216-76
California Institute of Technology



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