[Histonet] alternative fixative to PFA - HA tag detection

[Sebastien Thuault]

Hi,

 

We have been trying to detect an HA-tagged protein in mouse brain slices
using immunohistochemistry but we failed to detect any signal so far. There
was nothing there, zero.

We have tested the antibody in primary neuronal cultures and it works fine.
The antibody is a monoclonal from Covance (clone 16B12 , Number MMS-101P).
Does anybody have some experience with this antibody or with HA-tag
detection in brain slices? 

Our procedure is a standard immunohistology procedure that works with many
antibodies. We perfuse the brain with PFA 4% in PBS and then store the
tissue overnight in PFA 4%. We cut 50 microns slices on a vibratome. The
secondary antibody is coupled to Alexa 568. We excite the dye with a laser.
Recently we tried to apply the exact same protocol we use for the cultures
to the mouse tissue, we fixed fresh frozen section cut on a cryostat with
PFA 4% for only a short amount of time (a few minutes instead of 24 hours or
so). When we did that we could detect some neurons labeled but very few, in
fact much fewer than expected which suggest that even with these
modifications the detection was far from optimal.

 

We think that the fixation method might be altering the antigenicity of the
HA tag, what are the other fixation method that we could try for these
staining? 

 

Any other suggestions?

 

Thanks for your help!

 

Sebastien Thuault, PhD

Columbia University