methanol vs paraformaldehyde fixation

Deana Leonard dmarie at gsbs3.gs.uth.tmc.edu
Wed Sep 26 11:04:20 EST 2001


Hello- 

I have a question about paraformaldehyde fixation vs. methanol fixation of
TC cells. I'm trying to subsequently immuno-stain with my affinity purify
antibody. The problem I'm having is with the two fixation protocols I get
entirely different results.

With the paraformaldehyde fixation my protein appears to be excluded from
the nucleus except in early prophase of the cell cycle (when the DNA is
condensed) it is in the nucleus.

BUT

staining after Methanol/acetone fixation shows exclusively nuclear staining.
I'm stumped how to determine which is the correct localization of my
protein. Is one method more reliable than the other? From what I've read
paraformaldehyde is considered to be more reliable. Does every one agree?

I've stained four different cell lines and they all give the above
discrepancy based on fixation method. My protein does not express well in
cells- I've tried making a GFP-tagged construct and a HISMAX tagged
construct with no luck getting noticeable expression.

I'm using 4% paraformaldehyde for 30 minutes followed by 15 minutes of
triton extraction buffer (0.5 % triton X) to permebolize the cells.

For the methanol fixation I'm using 1:1 methanol to acetone for 10 minutes.

All done at room temperature.

Does anyone have any advice?
Thanks,
Deana 






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