[Cell-biology] IHC CD4 and CD8 staining
(by kcarbaja from uci.edu)
Tue Sep 16 17:08:05 EST 2008
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
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
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