Paraformaldehyde and autofluorescence.

Dave Peritt peritt at
Tue Mar 4 13:10:15 EST 1997

In article <l03010d00af40f5cdc615@[]> IMMUNO-SHERBROOKE,
immunolo at COURRIER.USHERB.CA writes:
>	I perform immunophenotyping of human lymphocytes by flow cytometry.
>I use 1% paraformaldehyde solution (PFA; Sigma) in PBS for cell fixation.
>The autofluorescence of fixed cell exited at 488nm is increased 6-7 fold
>comparing to nonfixed control.
>	1. Can someone suggest me an alternative PFA source which gives
>minimal autofluorescence?
>	2. Can someone suggest me a post-fixation protocol which reduces
>PFA-fixed cell autofluorescence?

are you fixing before or after you stain the cells??  You should be
fixing after all staining is finished otherwise the Ab may get into the
cell and the intracellular environment is much more sticky... Post
stained fixed cells are good for at least several days in the dark and at

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