Just some further comments re: ICC protocols
I agree with most of what William Sun says and here are my variations
on the theme.
1) 3x 10 minute washes should be sufficient between primary, secondary,
the aviden/biotin (ABC) step and the chromagen (often DAB) step.
2) perfusion with 4% paraformaldehyde with post fixation overnight is
suitable for most antigen in my hands. This can be variable and
crosslinking fixatives such as paraform. can be troblesome. Play
around with the fix time. Another option is to use a non-crosslinking
fixative. We have had good success with this technique for some
antigens. Currently we are using "Histochoice" fixative from AMRESCO.
No, I don't work for them or own stock. This fix also works well for
preserving RNA for in-situ.
3) I use 0.3% Triton X-100/2% normal serum from the animal that the
secondary antibody is made in/ and sometimes 2% BSA as a diluent for my
primary incubation and for the secondary incubation.
Primary: 4 hours at RT or overnight at 4 deg. C.
Secondary: 1 hour at RT
ABC: made up in only PBS and done for 1 hour at RT
Blocking with the above diluent only can also help clean up things.
try for 30 min just prior to the primary antibody step with no rinses
4) Your can post fix overnight and cryoprotect by adding sucrose to the
post fix solution. (done at 4 deg. C.)
5) Sometimes you can enhance results by preincubating your tissue
sections in DAB for 5 minutes and then placing them in a fresh DAB
solution containing the necessary H2O2.
6) preincubating the primary antibody in normal sera of the animal type
that the tissue is taken from can help remove some cross reacting IgG's.
7) fixation of the tissue sections on the subed slides serves only to
crosslink the sections to the gelatin materiel on the slide and indeed
be minimal. If sections fall off then try a little more. Hint: try the
charged glass slides from Fisher called "superfrost Plus". No subbing
necessary! We have had good luck with these and many pathology depts.
are known to use them.
8) If there is a lot of non-specific background one source could be
endogenous peroxidase activity (especially if you see a lot of DAB
staining in red blood cells). The cure for this is a 10-20 minute
incubation in a solution of 0.3% to 1% solution of H2O2 in PBS with
2x 10 minute washes afterwards.
Hope some of this helps. Good luck. figuring out ICC protocols can be
a pain sometimes. Feel free to e-mail for details on any of this if you
Jerry Clayton, PhD
Dept. Neurology, UCHSC
claytonj at essex.hsc.colorado.eduioana at iastate.edu (ioana sonea) wrote:
>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.