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To demonstrate Ki-67, it seems that fixation of adherent cells with -20 C
methanol is sufficent. Following fixation and a blocking step of PBS/BSA
for one hour, nucleolar staining of this antigen using an indirect
immunofluorescence technique (anti Ki-67 for one hour, brief wash, goat
anti MsIg-FITC for one hour and another brief wash) gives beautiful
results. No other permeabilization step seems to be required. However,
using anti p53 (as well as one other nucleus/nuclear antigen-specific
antibody, i.e.,
anti RNA polymerase I) we have not been able to demonstrate strong nuclear
staining but rather find the staining most strongly in the cytoplasm.
My question: Is it possible to demonstrate nuclear antigens with antibody
on fixed cells using only a -20 C methanol fixation step, or is it
necessary to include a second step with, e.g., Triton X-100?
Thanks for any help from the group.
Ray
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Raymond B. Hester "We want only to show you
Flow Cytometry Lab something we have seen and to tell
The University of South Alabama you something we have heard...
Mobile, AL 36688-0002 that here and there in the world
Email: rhester@jaguar1.usouthal.edu and now and then in ourselves
Voice: (205)460-6029 is a New Creation."
- Paul Tillich, The New Being
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