IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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!!



More information about the Neur-sci mailing list

Send comments to us at biosci-help [At] net.bio.net