Immunohistochemistry - Leakage of protein from nucleus?

Emir Khatipov khatipovNO at NOuchicago.edu
Thu Oct 4 18:20:33 EST 2001


Melissa,

I recommend paraformaldehyde (PFA) fixation:
3% PFA/2% sucrose in PBS for 5 min at R/T, and wash with PBS 2-3 times. PFA,
as well as formaldehyde, will easily penetrate cells and crosslink all the
proteins, as well as crosslinks cells to collagen, poly-L-Lys or whatever
else you might want to use for coating the slides.

Dissolve PFA at ~65C and add ~150ul of 1N NaOH or it will never get into
solution : 1) heat PBS to 65C in a water bath, 2) add PFA, 3) add NaOH, wait
until PFA dissolves completely (~30-60 min) 4) add sucrose, 6) cool down to
R/T and adjust pH to 7.0 with 1N HCl. Use within a week.

Tell me if it worked for you as much as it works for me.

Emir Khatipov
University of Chicago

"Melissa Greeve" <gliftor at hotmail.com> wrote in message
news:350b1ac.0110040044.4f1d3f8e at posting.google.com...
> Hi everyone again,
>
> Thanks for your replies to my last posting of immunoprecipitation
> woes. I seem to have managed to get IPs to work, resorting to the use
> of a more dense SDS-PAGE gel to avoid the problem of the protein G
> band - I have, however, yet to get consistent results. But it's only a
> matter of time, more sleep deprivation and some more tweaking... :)
>
> Anyway, onto my next curious problem. One of the important experiments
> that will (hopefully) nicely supplement the IP results will be
> immunohistochemistry of cells grown on slides. The protocol itself
> appears to be fine, but one of the proteins I have been staining for
> is meant to be (published for other cell types) nuclear - but I have
> only been able to show that it is cytoplasmic and perinuclear. So far,
> I have tried two different antibodies to the protein. In contrast, I
> have shown that another protein, also published as nuclear, is indeed
> expressed in the nucleus and cytoplasm in a cell-cycle dependent
> manner.
>
> Someone has suggested to me that one of the potential reasons for this
> discrepancy could be leakage of the protein from the nucleus into the
> surrounding cytoplasm during insufficient fixing of the slide. So, I
> tried chilled acetone, with nasty results - the cells, understandably,
> dehydrated.
>
> Is this a logical explanation? Why would one protein act like this
> when fixed in 95% ethanol and not the other (both have similar
> cellular functions)? Can a PhD get any more complicated??
>
> I will try a different cell type as a control, to confirm that the
> apparent localisation of the protein in cytoplasm results from
> insufficient fixing, otherwise, maybe my cell type is a strange
> exception! It's important to demonstrate the true localisation of this
> protein before I obtain any results (of course, analysis of this
> protein is crucial to my project!)
>
> Thanks again,
> Melissa Greeve
> PhD Student
> University of Western Australia





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