Hi!  I currently do alot of alpha smc actin in mouse tissue with very
little background.  So far I have worked exclusively with heart and arterial
tissue.  I would be more than happy to fax along my protocols. I should mention
that I have different antibody titrations depending on which fixative I use.
Also, I incubate the primary, secondary, and enzyme at 4 C which has helped to
eliminate much of my background problem.  I have also included a high salt
buffer rinse after application of the primary, secondary and enzyme in addition
to my PBS rinse.  I'm not sure how much of a difference this makes but my
staining is good with little background.  Finally I generally develop
(microscopically) only 3 to 4 slides at a time so I can monitor the staining and
background and stop the substrate reaction when the staining is good and the
background has not yet come out!  My processing protocol is only 3 hours, very
abbreviated, and generally I only fix the tissue for 3 hours at 4 C.  You may
have to adjust the titrations or conditions in your laboratory but my protocols
may be a good starting point.  Please send your fax number if you would like
further information!


Jennifer Hoover
Novartis Pharmaceuticals