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- From your description I assume that you want to label cell surface antigens
and not intracellular antigens in the lymphocytes. If this is so, I will
not suggest to fix the cells prior to immunofluorescence labeling, because
that would raise the nonspecific binding markedly. I have done
immunofluorescence labeling followed by FISH on breast cancer cell lines
(manuscript in preparation) and my suggestions are the following.
Perform the immunofluorescence labeling on live cells, fix them afterwards
very gently i.e. 0.5 % formaldehyde solution in PBS for 30 min at room
temperature. (Personally I prefer formaldehyde to paraformaldehyde solution
because the preparation of the latter one is such a hassle and I could not
see any differences between the two solutions in influencing the
autofluorescence during fixation.) Then the flow analysis (sorting) can
be performed. If you want to avoid the proteolytic treatment, the sorted
cells can be exposed hypotonic solution (the same way as chromosomes are
prepared from PHA stimulated lymphocytes), then Carnoy fixed, dropped on
slides, airdried and processed according to the FISH protocol. (Because of
the very mild fixation cells behave almost the same way as live cells
during the hypotonic shock.)
Regarding the alcohol fixation: The alcohol will not aggregate the cells,
the DNA released from damaged cell is responsible for the aggregation.
There is an old method to prevent this type of aggregation, application of
DNase enzyme on live cells prior to ethanol or methanol fixation drastically
decreases cell aggregation (Meth. Cell Biol. 1975, Vol 9. p 179).
I hope this will help.
Regards,
Janos
Janos Szollosi
Department of Biophysics
Medical University School of Debrecen
Nagyerdei krt. 98
H-4010 Debrecen,
HUNGARY
Phone/FAX: (36)(52)-412-623
E-mail: szollo@jaguar.dote.hu
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