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I am a friend of your friend, who had asked you my questions about PCNA. Thank
you for addressing my concerns on the bulletin board. I would like to clarify
my situation.
First of all, I am using unconjugated PCNA(PC10) and GAM FITC on frozen rat
liver. I am using detergent extraction with a lysing buffer consisting of BSA
and 0.5% Triton X-100 before fixation with methanol .This allows elimination of
the nucleoplasmic population of cyclin, therefore leaving cyclin that is
associated with nuclear structure (Bravo, 1987). I find that in staining
liver treated with various cell proliferation reagents, I get a very low
percentage(1-5%) of positive cells (brightly staining). This correlates with my
S phase estimates using PI. However, compared to the isotypic control, whose
protein concentration is matched, my PCNA stained samples exhibit a slight
shift in fluorescence intensity above the control . This happens more so in 4N
and 8N nuclei compared to 2N. My question is, could this be true staining or
should it be disregarded as background? Has anyone done similiar work and if so
do you get similiar results?
Thanks in advance,
Cheryl Torretto
torretc@aa.wl.com
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