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!!