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I apologize for the curt answer to your question a couple
days ago. Here is a more complete, hopefully better
answer.
You might try formaldehyde followed by MeOH as described by
in Cytometry 13:48-59 and/or 13:432-434. If this looks
promising, you should titer the formaldehyde concentration
against whatever assay seems appropriate - in your case
it would presumably be both the level of p53 staining and
the cyoplasmic/nuclear location ratio. An example of
titering the formaldehyde is shown in
Bauer & Jacobberger, 1994, Analysis of intracellular
proteins. in Methods in Cell Biology: Flow Cytometry,
Vol 41, eds. Z. Darzynkiewicz, H.A. Crissman, J.P.
Robinson, Academic Press, Inc., NY, pp 351-376.
An original article showing the same thing with deter-
gent permeabilization is Mann et al, 1987, Cytometry
8:509-517.
If you are not already, you should be aware of two
papers describing cytoplasmic p53 staining. These
are:
Shaulsky et al, 1990, Oncogene 5:1707-1711
Gannon and Lane, 1991, Nature 349:802-805
The following is a list of some reviews on the subject
of intracellular staining for quantitative analysis
of which I'm aware. There are others. The subject is
more complex than your original question and my
original answer. The original list was truncated.
Jacobberger JW, 1991, Intracellular antigen staining:
quantitative immunofluorescence. Methods 2:207-218
Lehman JM and Jacobberger JW, 1990, Analysis of viral
effects of cells utilizing flow cytometry. in "Flow
Cytometry and Sorting", 2cnd ed., eds. Melamed MR,
Lindmo T, Mendelsohn ML, Wiley-Liss, Inc., NY, pp623-
631
Jacobberger JW, 1989, Cell cycle expression of nuclear
proteins. in "Flow Cytometry: Advanced Research and
Applications", vol. I, ed. A. Yen, CRC Press, pp305-
326.
Clevenger CV and Shankey TV, 1993, Cytochemistry II:
Immunofluorescence measurement of Intracellular
Antigens. in Clinical Flow Cytometry, Principles
and Applications. eds. KD Bauer, RE Duque, TV
Shankey, Williams & Wilkens, Baltimore, pp157-176
Shapiro H, 1995, Practical Flow Cytometry. Wiley-Liss,
NY, pp248-250
For getting directly to some methods...
Handbook of Flow Cytometry Methods, ed. JP Robinson,
Wiley-Liss, NY, pp87, 118, 120
-- James W. Jacobberger Case Western Reserve Univ. 216-368-4645 (voice) 216-368-3432 (FAX)
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