[Histonet] anti-CD45 antibody for mouse parrafin sections

[Gayle Callis]

I suggest you buy the rat antiMouse anti CD45 monoclonal, the one you are
attempting to work with detects CD45 in rat tissues.  BD Pharmingen carries
the rat antiMouse.  I am not sure if the OX-1 clone even cross reacts to
mouse antigens, plus you will have mouse to mouse problems.  Any clone with
OX-1 is usually a rat clone.  

At 04:14 PM 4/29/2004 +0200, you wrote:
>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)
>
>
>
>
>
>
>
>-- 
>----------------------------
>*- mkempen -*
>MAILTO:m.vankempen <@t> erasmusmc.nl
>-----------------------------------------
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)