[Histonet] RE: Cell permeabilization and cell surface staining
Sometimes theory and practice doesn't match with each other at all!
That keeps us awake and makes our life both frustrating and
interesting! I am afraid that the situations as you sketched are not
that black-and-white in real life. For example there is a good change
that the cells got a bit damaged, allowing the antibodies go in and
out and having your internal proteins nicely stained. We once saw a
difference between a cytospin and cells that were attached to glass
(using the good old BioRad slides). The attachment procedure appeared
to be more gentle to the cells than spinning them down with a great
force, causing damaged cells at least at molecular level.
Furthermore there might be a molecular reason why the internal antigen
stained without Triton X100 treatment. Recently, Dick Dapson showed in
JOH that formalin fixation followed by alcohol dehydration may even
flip certain macromolecules inside-out. If this happens with your
'internal' protein you will get nice staining without the Triton X100
Why the Triton X100 treatment decreased your surface staining is
difficult to understand. First thing that came up with me is that your
antigen may shed from the membrane due to the Triton X100 treatment.
Hope this helps a bit.
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
NL-1105 AZ Amsterdam
Date: Thu, 30 Nov 2006 10:27:57 -0000
From: "Martin S."
Subject: [Histonet] Cell permeabilization and cell surface staining
I have been staining BMDC's to look at proteins which are present on
cell surface and in the cytoplasm. I fix the cells with 4%
paraformaldehyde in PBS, 20min on ice and permeabilize with 0.2%
X100 in PBS for 20min at room temp. My two problems are:
1) When I dont permeabilize the cells I can still detect internal
antigen with my primary antibody (cells are fixed 4% PFA, washed PBS
blocked with 2% BSA before incubation with antibody)
2) When I permeabilize the cells I get a decrease in the cell surface
Any suggestions greatly appreciated.
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