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I can't recall the reference for this info. but, I do recall reading
in a flow cyt. textbook, several of the answers to your questions. You
should definitely fix your cells AFTER antibody staining. The antibodies
you are using, I can almost guarantee, were generated with unfixed cells
(hence, the antigens were in their natural state). It has been very
clearly documented that fixing with paraformaldehyde can cause some
conformational changes in proteins. If you stain prior to fixing, the
Ag-Ab complex seems to be protected from these potential shape changes.
However, if you stain after fixing, the Ag you want to stain could
theoretically undergo a minor shape change, causing the Ab ligand to
either stain less intensely or not at all.
In our lab, we fix our cells using a 1% paraformaldehyde solution
(i.e. it's 1% prior to adding to the tube containing the cells to be
fixed). We usually use 1 mL of this solution to fix 1 million cells
(after spinning down and dumping supernatant). This fixing procedure
seems to work well; we've had no problems.
My regards,
Byram W. Bridle
U. of Guelph
Guelph, Ontario, Canada
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