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immunohistochemistry protocol

Annick Ansselin annick at cortex.physiol.su.oz.au
Tue Jul 25 03:08:17 EST 1995


In <3uhhtg$ovs at news.iastate.edu> ioana at iastate.edu (ioana sonea) writes:

>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!!

You could try blocking with rat serum before the antibody labelling.
It worked for peripheral nerve.



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