[Histonet] Fixing cells on coverslips for immunos

From:Mikael Niku

Dear Histonetters,

a basic question: how to make cultured cells stay on coverslips for 
immufluorescence staining?

We are culturing cells on lysine-coated glass coverslips and trying to 
immunostain them. We know the antibody works with acetone fixation and 
probably not with PFA (at least not in the case of tissue sections). The 
problem is, most of cells drop off during the staining. What can we do? 
I have done mainly tissue sections, so I'm in trouble with this one.

Currently we have tried like this: cells were washed with PBS, 
transferred to acetone for 10 min at +4C, back to PBS, followed with a 
very basic indirect immunofluorescence staining (with 30-minute antibody 
incubations, whole procedure within one day), and Faramount embedding. 
This works well for cytospin preps (the difference is that the 
cytospins, of course, are dried before the fixation; and we use 
Superfrost slides for them).

Is it possible (or even common) that an antibody might work with cell 
culture samples using a quick PFA (paraformaldehyde) fixation, although 
it only works with non-crosslinking fixatives for tissue sections?

With best regards,


  Mikael Niku             URL: www.helsinki.fi/~mniku/
  University of Helsinki  Dept. Basic Veterinary Sciences

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  Minusta se olisi erinomainen ajatus!                                                        
                                          - Gandhi


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