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immunohistochemistry protocol

ioana sonea ioana at iastate.edu
Tue Jul 18 18:59:44 EST 1995

In article <3u75sr$87k at neuro.usc.edu>, william at neuro.usc.edu (William Sun) says:
>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

Beyond mentioning that it seems excessively complicated,
I won't comment on your fixation protocol which may be required for
your antigen (4% PF works fine for most things if not cell surface antigens),
but the washes may be too little : we usually
wash 10x (5-10min each) after the primary, and 4-6x between primary and 
secondary, and secondary and ABC step. Should be the same for other substrates
(are you using a peroxidase-labelled secondary).
 Also how long is your incubation,
and at what temp? You could try incubating at 4C for 48-72 hours, or 37C 
overnight to increase specific binding.
Finally, one thing that will lead to blotchy sections is drying out at any
time of the tissue prior to final reaction.
Good luck!!

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