[Histonet] T cell staining in Spleen

[Felix.Rintelen <@t> serono.com]

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
immunohistochemistry:
Rat 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
F-6258)

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 4°C 10', dry 15' and then store at -80°C 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
PBS)
Wash 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

-------------

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



-----------------------------------------
S - This message contains confidential information and is intended only for
the individual named. If you are not the named addressee, you should not
disseminate, distribute or copy this e-mail. Please notify the sender
immediately by e-mail if you have received this e-mail by mistake and
delete this e-mail from your system.  e-mail transmission cannot be
guaranteed to be secure or error-free as information could be intercepted,
corrupted, lost, destroyed, arrive late or incomplete, or contain malware.
The presence of this disclaimer is not a proof that it was originated at
Serono International S.A. or one of its affiliates. Serono International
S.A and its affiliates therefore do not accept liability for any errors or
omissions in the content of this message, which arise as a result of e-mail
transmission. If verification is required, please request a hard-copy
version. Serono International SA, 15bis Chemin Des Mines, Geneva,
Switzerland, www.serono.com.