[Cell-biology] IHC CD4 and CD8 staining

[Kevin Carbajal]

Hello,I'm trying to stain for CD4 and CD8 antigens in mouse spinal cords
(that have vast T-cell infiltration) and can't get it to work.

My tissue is frozen and I have used it successfully in staining many other
antigens, but these don't show.  My primaries are rat anti-mouse antibodies
from Pharmingen (for IHC) and I have used two secondaries: a donkey anti-rat
PE, and a biotinylated rabbit anti-rat. I've fixed the tissues with 4%
Paraformaldehyde for 20 min, and with acetone at -20C for 10 min.  Blocked
with 5-10% of the corresponding serum and followed standard protocols
(washing, mounting, DAB staining in the case of the biotin. ab, etc).

My incubations are over-night in 4C for the primaries and 1.5 hrs RT for the
secondaries.
I've used diverse concentrations of the antibodies.  I have also tried it
all on spleen sections for positive controls.

What could I be doing wrong?  Do I need to buy new antibodies?
Thank you for any suggestions!
-- 
Kevin S. Carbajal