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