mouse CD4 and CD8 on paraffin

From:Jan Bauer

Dear collegues,

I know that the question of staining of CD4/CD8 in paraffin-embedded mouse tissues has been asked several times during the last years. I am desperately looking for a CD8 antibody which works in mice. Until now we have tried antibodies from Serotec (KT15), BD Pharmingen (53-6.7) and Santa Cruz (sc7979, clone D-9) and none of them worked. Regarding the SC antibody: this one stained macrophages and dendritic cells but certainly not CD8 lymphocytes, although the datasheet implied that it should work on paraffin.  We have tried this with several pre-treatments including heating with microwave or steaming device (1 hr.), trypsin, protease and in addition tyramid-biotin enhancement. Recently the work from Whitefield et al., J Histochem Cytochem; 43: 313-320 (1995) came to my attention and they describe staining for CD4 and CD8 on periodate-lysine-paraformaldehyde fixed, paraffin-embedded material. The pictures they show are very convincing but I still have soem doubt. I know and used this fixation technique and can understand that this might increase the number of epitopes free for staining with the antibodies. It however is hard to believe that there are so much more epitopes that it results in a staining which is not possible with 4% paraformaldehyde and intensive enhancement with the biotin-tyramid system (with this we generally can dilude antibodies 10 times as high).  So, coming to my question: Is there someone who has been able to verify this staining of CD4 and CD8 in mouse material? The Whiteland group uses incredibly low dilutions for some antibodies  (undiluted for KT15, W3/25, 1:5 for OX42 and 1:20 for CD4 (RM4-5). Can this make the difference?

Thanks in advance for your commentaries!
Jan

-----------------------------------------------------
Jan Bauer, Ph.D
Div. of Neuroimmunology
Brain Research Institute
Spitalgasse 4
A-1090 Vienna
Austria

tel:    +43-1-4277 62813
fax:    +43-1-4277 9628


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