[Histonet] IFA staining problems

From:Christopher C Overend

I am new to the technique of immunofluorescence, and have been doing some work with it lately on cultured cells. Specifically, I have been staining intracellular virus with very good results. However, when i try to detect cellular proteins, I do not get any staining. The proceedure i have been using consists of "fixation" with 90% acetone for 30 min, at 4degrees C. the rest of the process had been much like an ELISA, all incubations have been for 30 min at 4degrees. The buffer i have been using is PBS, 1%BSA, 0.1%NaN3. Someone mentioned the problem could be from the acetone and suggested trying a glyoxal fixative. Can anyone offer some insights, or if there is a different protocol i might have to follow using a glyoxal fixative with tissue culture?
Thank you!

Christopher Overend
Ph.D Student
University of Connecticut
Department of Pathobiology and Veterinary Sciences
Storrs, CT


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