Heya,
I've been having some profound difficulties in performing anti-BrdU
immunohistochemistry on paraformaldehyde-fixed cells. The
paraformaldehyde-fixation is, unfortunately, a necessary step in that my
cells of interest have been fully differentiated and will not remain
adhered to the substratum when other fixatives (methanol, acidic-ethanol)
are used. Fixing with paraformaldehyde is the ONLY thing that keeps them
on the bottom throughout the whole process. The problem is, it looks like
the paraformaldehyde-fixing also masks the antigen from the antibody.
I'm hoping this is a difficulty with the antibody. I used a
different cell line which sticks really well (such that
paraformaldehyde-fixation isn't mandatory) to test a couple of fixation
conditions, side-by-side:
- when the adherent cells are "fixed" with simply acidic-ethanol, I can
detect BrdU incorportation easily with my anti-BrdU antibody.
but
- when the adherent cells are fixed with paraformaldehyde (2%) and
post-fixed with methanol, I can no longer detect incorporation -- and the
ONLY difference is the fixation. I use the same antibody dilutions, the
same concentrations of DNase I, etc, on all the cells.
So, I was hoping someone out there who does this kind of experiment could
offer some advice? Is it possible that the particular anti-BrdU I am
using cannot work in paraformaldehyde-fixed tissue.....but......a
different anti-BrdU antibody could work?
I have seen reports in the literature of BrdU immunohistochemistry on
paraformaldehyde-fixed tissue, so I'm sure it can be done. I'm just at a
loss to figure out what to do here -- I have to keep using
paraformaldehyde or else my differentiated cells will drift off.
Does anyone have any better antibodies (or ideas)?
John
john.hines at yale.edu
P.S.: I am already plating the cells onto fibronecting-coated dishes.
This is by far the best coating matrix (much better than poly-L-lysine or
collagen). Even so, I need the paraformaldehyde to KEEP the cells stuck.
