antibody staining on slides

Tali Melkman melkman at brandeis.edu
Fri Oct 10 01:39:22 EST 2003


Hi!
I am trying to stain both adults and embryos on polylysine coated 
slides. I have been putting adults on coated slides and pressing down 
slightly such that they burst open at the vulva releasing their eggs. 
Then I permeabilize the animals by freeze cracking.
My first problem is that the eggs then sometimes stick to the slides 
but the adults most times don't. How do I get the animals to stay on 
the slide?
The second problem is that I have high nuclear background that 
fluoresces orange on the GFP filter, but has nothing to do with the 
fluorophore the secondary antibody is conjugated to. This background 
then interferes with staining visualized with FITC. What is this 
background? how do I get rid of it?
  I would appreciate any suggestions/ideas.
Thanks in advance,
Tali

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