[Histonet] T cell staining in Spleen


Dear all,

I'm trying to stain CD4 and CD8 T cells in cryosections from mouse spleen.
However, with not much success so far. The good thing is that I only see
staining in spleen (and not in liver, which was embedded in the same block
together with the spleen). However, I don't really get staining in the
areas where I would expect to see it and I only see small clusters of cells
instead of big ones. And the real problem is that I get the exact same
staining with the Isotype control as with the specific anti-CD4 and
anti-CD8 antibodies. (I tired to post a picture of the staining on
www.histonet.org, but so far I always get a message back of delivery
failure, but I will try again...). Somehow it seems that IgG2a antibodies
unspecifically bind to spleen but not to liver...

I'm using the following antibodies that are supposed to work  in
=0ARat anti-mouse CD4 IgG2a, Clone RM4-5, BD 550280
Rat anti-mouse CD8a IgG2a, Clone 53-6.7, BD 550281
Rat IgG2a Isotype control Clone R35-95, BD 559073

Secondary Antibody was Goat anti-Rat IgG (whole molecule) - FITC (SIGMA

The protocol that I was using is the following:

Sections: 10um of mouse liver & spleen embedded in OCT medium
Dry over night at room temperature
Fix in 100% Aceton 4C 10', dry 15' and then store at -80C until use

Take out of freezer and dry 5'
Rehydrate 20' in PBS
Block 20' with 3% normal goat serum in buffer (0.5%BSA, 0.1% Tween20 in
=0AWash 5' in buffer
Incubate with 1 Antibody (see above) 1hour (Dilutions: 1:10, 1:25, 1:50,
Isotype control 1:10 and 1:50)
Wash 3x 5' with buffer
Incubate with 2 Antibody (see above) 1 hour (Dilution 1:25)
Wash 3x 5' with buffer
Mount in Fluoromount G (Electon Microscopy Sciences)

I also tried to do Immunohistochemistry with the same antibodies using
Vectastain Elite ABC Kit (Rat IgG) (Vector Laboratories) Peroxidase system
with NovaRed as substrate and I get a similar result as with the
fluorescence staining.

I would very much appreciate your advice or to hear your opinion. Maybe
somebody may recommend another protocol / antibodies that should work on
cryostat sections.

Thank you very much in advance,

Best Regards,



Felix Rintelen (Post Doc Fellow)
Serono Pharmaceutical Research Institute
=0A14, Chemin des Aulx
1228 Plan-les-Ouates
Geneva, Switzerland=0D

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