We have recently established a protocol to grow a primary culture that works very well. Unfortunately, we have problems staining these cultures. We need to fix them with a very low % of paraformaldehyde (1% PFA) to obtain good staining for some of our crucial antibodies, but with such conditions the cultures come off the coverslip when rinsed to many times. We have tried coating our coverslips (glass and plastic) with poly-L-lysine and/or collagen. The only way the cells stay on the coverslips is if we fix them with 4% PFA or methanol, but then the staining is awfull!?! Can anyone help unsolve this mystery?
Primary cell fixing problem
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