[Histonet] RE: need help with IHC detection of human cells in mouse tissue

From:"PALMER Jason (SVHM)"

I have used a mouse anti human mitochondria antibody from Calbiochem before to successfully identify a variety of human cells in mouse background in paraffin embedded tissue. I am pretty sure, however, that they don't manufacture this one anymore, but I wouldn't be surprised if other companies are producing other human-specific mitochondrial Abs.

Jason Palmer

Bernard O'Brien Institute of Microsurgery 
42 Fitzroy St, Fitzroy Victoria 3065 
tel +61 3 9288 4018 
fax +61 3 9416 0926 
email: palmerj@svhm.org.au 


I was wondering if anybody could help me out.  We want to look for human cells engrafted in mice, particularly in formalin-fixed, acid decalcified, paraffin-embedded tissue.  I have so far tried two anti-human beta-2 microglobulin antibodies.  

The first I tried has been used in human/sheep transplant tissue, and I tried it, but unfortunately it is an IgM kappa, so it failed with the Biogenex mouse-on-mouse IHC kit, which it turns out is not made for IgM antibodies.  If anybody is aware of a good way to use a mouse IgM on both human and mouse tissue, I'd love to know.  I have looked for a good secondary antibody for this purpose, but have had no luck so far.

The second antibody I tried is from Novocastra and distributed by Vector Labs.  It is a rabbit polyclonal to human beta-2-microglobulin.  The trouble I had was nonspecific binding in control mouse tissue.  I was thinking to use some of the Biogenex mouse-on-mouse kit reagents to help block mouse antigens, but I do not know if this would actually work.  The second problem with this antibody was that I did not always see positive marking in human tissue (especially osteocytes), which I thought should be pretty well all positive since it is human tissue.  Is the expression of beta-2-microglobulin so variable?  

Has anyone tried any other general human cell marker in other animals?  I did a quick search for beta actin, but it seems most of those cross-react with mouse.

Thank you!

John Garlits, M.S.
Senior Research Technician
Hematology Oncology Division
Experimental Hematology Department
St. Jude Children's Research Hospital
332 N Lauderdale
Memphis, TN 38108

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