Immunostaining of nuclear proteins

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Fri Nov 5 12:22:23 EST 1999



Stephen Snowdy wrote:

> Does anyone know if methanol does a good job of permeabilizing adherent
> cells all the way down to the nucleus?  Are any special steps necessary
> to immunostain epitopes inside the nucleus?
> --
> Stephen Snowdy
> Department of Pharmacology
> College of Medicine
> University of North Carolina-Chapel Hill

Hi Stephen,
Many people working with nuclear proteins prefer fixation with
paraformaldehyde over fixation with organic solvents. That´s because
nuclear structures are preserved much better, and localization determined
by immunostaining appears to be more reliable. Methanol, aceton, or a 1:1
mixture of both can be used, but must be handled properly. That is:
solutions at least -20C cold, fixation in the freezer for the shortest
possible time (30 seconds is a reasonable start to play around and find
optimal conditions). Right after fixation, cells should be washed in,
e.g., PBS and inspected in a phase contrast microscope. Well fixed cells
should look quite like living cells.
If you want to try paraformaldehyde (which is definitely better in my
hands), remember that this stuff fixes the cells, but does not
permeabilize them. To this end, fixed cells should be briefly (3min)
treated with PBS+0.3% Triton X-100. After permeabilization, you can
(optinally) treat the cells for 30min with PBS+100mM glycine (to quench
residual crosslinking activity of the formaldehyde), and (necessary) block
them for 1h with, e.g.  PBS+3%BSA before incubaion with the primary
antibody.

Nuclei are notorious for being prone to unspecific binding of antibodies,
so is necessary to include good controls: several dilutions of the primary
and secondary antibody, secondary antibody alone, inhibition of the
"specific" signal by preincubation with the antigen (peptide or
recombinant protein).

Hope this helps,
Frank





More information about the Methods mailing list