fixation with FITC and PI

Wolfgang Schechinger hubahopp at
Thu Nov 21 10:23:32 EST 2002


I remember vaguely that I incubated cells with a fluorescein derivative
from which fluorescein is liberated by an ezymatic reaction and that I
quenched the rxn with formaldehyde to kill the cells. I think it worked.

If you tend to use glutaraldehyde and then do the FACS, you should see the
background also in your controls and thus be able to subtract it
appropriately. There are some protocols out that utilize sodium borohydride
or cyanoborohydride to quench background fluorescence in immunocyto- and
histochemistry, you might adapt them for your need, if necessary.
Maybe shaking G-CHO with active charcaol and filtration before usage also
might do te job.

The few percent (or less) of methanol in your formaldehyde fixative
shouldn't kill the fluorescence actually (unless you use 50% MeOH or more).
If you want to be sure, prepare a fresh solution of (solid) paraformaldehyde.




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