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If you analyze within 2 hours of fixation with 0.5% PFA, then you can use PI:
do standard PI staining, wash, then fix. If you wait more than 2 hours, the
signal really degrades.
The other alternative (which we have used for combining with intracellular
staining) is to use EMA (ethdium monoazide). Stain cells with 5 ug/ml in the
dark (20 min), wash twice, then expose to a UV source (like a UV light, even I
think, a fluorescent light bulb). Wash. EMA is UV-crosslinked to become
covalently bound to the cells and lights up cells that were dead before fixation
and permeabilization.
The EMA was published by a couple of people; see the Molecular Probes catalog
for specific references.
mr
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web