[Histonet] IHC on Mouse EAE brain tissue (background)

From:Jamie E Erickson

Hi All,
            I'm hoping someone can help. I am staining mouse EAE brain 
frozen  tissues with BD biosciences CD45/B220 as well as there GL7 
(activated T+B cell marker) and have some background issues. The CD45 
/B220 looks good with minimal background and nice cell associated 
staining.   I had worked out these conditions on spleen for each antibody 
separately prior to running these brain samples. Briefly:

1.  I air dried  frozen brain sections overnight then  fix with acetone 
75%/alcohol 25% 5 minutes at RT then placed in buffer
 2. DAKO block H202 0.03% 10 minutes, (CD45), 5 minutes (GL7)  (WASH)
3. Streptavidin/biotin block (vector labs) 30 minutes each,(WASH)
4. serum block 10%donkey (CD45) 10% Horse (GL7) 15 minutes, (no wash)
5.  CD45 or GL7 (2.5ug/ml) 1 hour RT, (wash)
6. secondary for CD45 anti-Rat (fab)2(1:500) 30 minutes RT.(wash), for GL7 
anti-Rat IgM (1:200) for 30 minutes.
7. ready to use strepavidin peroxidase (vector labs ) 30 minutes RT.(wash)
8. DAB + DAKO 2-4 minutes.(wash,water)
9. Counterstain etc..

The problem is the CD45 staining has minimal background and the GL7 has 
much more on a serial section (could be peroxiade?). The background is a 
diffuse light brown staining over most of the section, Could this be due 
to the IgM pentameric antibody or something else I can block against?  The 
positive control spleens had positive staining and no background...very 
clean in both CD45 and GL7.  So I thought I was OK. Would adding 2.5% 
mouse serum to the block  and primary help?

 It seems to me something about the brain samples that is causing this 
background.  If anyone has experience background with brain IHC I'd 
appreciate any input you may have. 


Jamie Erickson
Sr. Research Associate 
Department: DSMP
Abbott Bioresearch Center
100 Research Drive
Worcester, MA 01605-4341
FAX: 508-793-4895
e-mail: jamie.erickson@abbott.com

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