RE: lacZ+ immunostaining
Aldehyde fixation will do nicely even for the immuno especially since you
state you'll be using an immuno amplification system (e.g. EnvisionTM). You
WILL need to develop the X-gal precipitate before the immuno step as fully
precipitated X-gal is stable to mild organic solvents (dehydration and
clearing post-HRP). This combination, blue-green X-gal and DAB),Donna
Physiology/Biophysics and Orthopaedic Surgery
University of Arkansas for Medical Sciences
should make a very pretty picture. Good luck,
From: Peter Huszthy [mailto:email@example.com]
Sent: Wednesday, April 17, 2002 10:52 AM
To: HistoNet Server
Subject: lacZ+ immunostaining
Good day to you, all histopathologists!
I have frozen sections of brain tissue, where I would like to show that lacZ
gene expression is localized to neurons.
Thus, I would like to perform both lacZ and immunostaining against
Neurofilament (DAKO mouse monoclonal) on the same section. Since the lacZ in
this case is localized to the nuclei, it would not overshadow the
Neurofilament staining, localized to axons.
For lacZ staining I have prevoiusly used 0.2% glutaraldehyde+ 2%
paraformaldehyde in PBS as fix.
For the immunostaining with this primary antibody, aceton was good, followed
by DAKO`s envision kit with HRP-labelled polymer and detection with
DAB+substrate-chromogen. How would I combine the two methods the best way?
Would the lacZ fix work all the way thru or would it probably destroy the
antigen? I have very few sections, so I cant do too much trial and error.
Thanks for your help. Peter
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