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immunohistochemistry protocol

William Sun william at neuro.usc.edu
Fri Jul 14 20:33:15 EST 1995

I am having problems getting good results doing immunohistochemistry on
rat brain sections.  Can anyone recommend a good, detailed protocol?

My protocol in brief:
-perfuse rat with saline and then 4% paraformaldehyde
-remove brain, wash in PBS
-place brain in PBS with 30% sucrose until it sinks
-freeze brain in isopentane and thaw rapidly in PBS
-freeze again, cut 50 um sections in cryostat, mount on subbed slides
-fix briefly (10 min) in paraformaldehyde, wash with PBS
-block with 20% horse serum, wash
-primary antibody in 10% horse serum
-wash 2X PBS, add secondary Ab in 10% horse serum
-wash 2X PBS, add DAB substrate.

I get alot of "spots" or regions with nonspecific staining.  Often the 
staining is very light.  I would appreciate any suggestions.

William Sun, Ph.D.                      Phone: (213)740-3406
Hedco Neuroscience Building             Fax: (213)740-5687
University of Southern California       Pager: (310)499-8670

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