[Histonet] anti-CD45 antibody for mouse parrafin sections

[m. van mkempen]

Hello,

Are there any people who have experience with the following anti-CD45
antibody: mouse monoclonal, clone OX-1 from BD #550566.

I am trying to get this antibody to work on MOUSE spleen parrafin
sections (It works in RAT cryosections). The tissue has been fixed in 4%
paraformaldehyde during 24h at 4 C. After making the sections (5-6
microns) and deparrafinizing I did the following steps:

Endogenous peroxidase blocking with 3% H2O2 in methanol for 20 min
Rinse with PBS
HIER with a citrate buffer (pH 6,0 and microwave 450W, 9 min)
Rinse with PBS
Block with PBS 5%BSA (30 min, RT)
Incubate with primairy antibody dilluted 1:50 in PBS 5%BSA at (O/N, 4 C)
Rinse with PBS
Incubate with rabbit anti-mouse immunoglbulins (DAKO #Z 0259) dilluted
1:50 in PBS 5%BSA (30min, RT)
Rinse with PBS
Incubate with monoclonal mouse PAP (DAKO #P 0850) dilluted 1:50 in PBS
5%BSA (30min, RT)
Rinse with PBS

Enzyme detection with DAB substrate (5-7 min)

Afterwards I can not detect anything besides some background from the
endogenous peroxidase. Should I try another antibody or can I improve my
protocol?

Best regards, Marion van Kempen (Erasmus MC, Rotterdam The Netherlands)







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