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Vince Shankey pointed out that isolated nuclei swell and may lose
antigens. This may require post-fixing. He also mentioned that
directly conjugated, FITC labeled antibodies may give better
labeling without such high backgrounds.
Karen Holdaway nicely summarized the various combinations of
fixation and permeabilization that are used.
paraformaldehyde, methanol, Tween 20
paraformaldehyde, lysolecithin or saponin, methanol
prarformaldehyde, Triton X-100
She implied that one must try various protocols to find the one
that works best with your cells and antigens.
Bruce Davis reported good results using Caltag's fix and perm
with several antigens.
Jim Jacobberger refered to an article by
Clevenger (Cytometry 1985) using formaldehyde and Triton X-100.
He points out that one must titrate both reagents to optimize
results for a specific antigen, and that proteins may remain
associated with each other thus "masking" them from the antbody.
He implies that adding methanol fixation may make some antigens
more available.
Visia Dragowska refered to an article in BioTechniques 19:192
(1955) that gives beautiful nuclei. She has had good results
with Caltag's fix and perm although there is more clumping with
nuclei than with whole cells. She says that using RNAase may
help reduce the background.
I hope this is helpful and that I haven't badly misquoted anyone.
Thank you all for your help!!
Ken Ault
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