Bruce,

Although I am not familiar specifically with activation of PKC gamma, many
of the signaling kinases such as MAPK / ERK, JNK, etc are activated by dual
 phosphorylation of specific nearby neighboring amino acids. I suggest you
consider looking into the availability of antibodies that will specifically
recognize the active (dual phosphorylated) form of PKC. I know Promega has
similar AntiACTIVE antibodies for ERK, JNK, etc. and New England Biolabs or
UBI may have similar antibodies.
Regarding prevention of evaporation, you might try the type of slides
containing incubation chamber (gaskets raising the coverslip up and seal
off the chamber) that is used for in situ hybridization.

Ty Lee

----------
From: Bruce A Rasmussen <brasmuss@osf1.gmu.edu>
To: HistoNet@Pathology.swmed.edu
Subject: Standards and Control for PKC IHC
Date: Monday, January 25, 1999 11:12 AM


Greetings histoneters,
I am going to attempt to image the distribution of PKC gamma in coronal
rat brain section (20 microns) using immunohistochemistry. The objective
is to get dissociative results between experimental and control animals
with a learning and memory paradigms. Is anyone aware of methods for
creating standards? I am leaning toward the technique of mounting sections
from both control and experimental groups on the same slides and pipetting
the incubating solution directly onto the tissue slices. Any suggestions
on solution volumes and methods to prevent the solution from evaporating?
The protocol calls for incubation with gentle agitation overnight at 4
degrees C. Absolute values for quantification would be gravy but the main
necessity is comparability between batches and antibody economy.
Thanks for your help
Bruce


--------------------------------------
Bruce Rasmussen
Predoc Fellow in Experimental Neuropsychology
The Krasnow Institute for Advanced Study
George Mason University
Mail Stop 2A1
Fairfax, VA 22030-4444
Office:703-993-4358
Lab:703-993-4369
Home:703-765-4570
Fax:703-993-4325
---------------------------------------