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suggestion - immunohistochemistry protocol

Andrew Ray aray at emory.edu
Sat Jul 15 22:21:31 EST 1995

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

>I am having problems getting good results doing immunohistochemistry on
>rat brain sections.  Can anyone recommend a good, detailed protocol?
>I get alot of "spots" or regions with nonspecific staining.  Often the 
>staining is very light.  I would appreciate any suggestions.
Along with the addition of Triton X (especially since 50 um sections are rather
thick, you could try several of the following:
1.  Cut back on the second fixation (post-cutting).  The light staining could
be due to over-fixation.  We fix just during the perfusion and get good 
2.  Cut back on the % of serum used for the blocking step.  It may or may not
be high enough to block some specific staining, too.
3.  What kind of secondary do you use?  You might try to enhance the staining
by using a biotinylated secondary and ABC-peroxidase protocols, or use a PAP
tertiary.  Either of these should enhance specific, but not nonspecific, 
4.  Try Ni intensifying your DAB reaction, but be warned - this can increase
background labelling as well!
5.  I don't know what type of primary you're using but you can try to increase
the concentration (although you probably already tried this).  
6.  You may just have a poorly binding primary (I've had one or two myself) or
a low antibody titer - especially if it's "homemade."
As far as the regional nonspecific staining, the only things I can suggest are:
1.  Reduce the secondary Ab concentration.
2.  Try a different blocking serum.  That is, try a combination.  For instance,
we've found that using 4% rabbit serum and 4% human serum works better than 8%
rabbit serum when blocking monkey tissue.
3.  Make sure that your slides are getting good coverage, and if you're 
reacting in jars, make sure there is good flow over the slides.  Put them on a
shaker if necessary, or speed it up.

I hope these help.  Good luck and let me know if any of this is useful.

Andrew Ray
Emory University Dep't. of Neuroscience
aray at emory.edu 

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