Immunohistochemistry HELP--paraffin vs frozen sections

[wetheril]

Hi,

I'm new to immunohistochemistry slide staining, and I'm having problems getting the staining to show correctly. So far, I've been staining lymph node tissue for CD62L (L-selectin) and CD3, both of which are supposed to be quite abundant on the surface of regular T cells in the lymph node (this is a control).

First, I tried to stain paraffin sections using a protocol given to me by another lab member who's done some staining before (the protocol is not very specific). I stained with the following primary antibodies:

CD62L by GeneTex - Cat # GTX11364
CD62L by BD Bioscience Pharmingen - Cat # 550397
CD3 by BD Bioscience Pharmingen - Cat # 550367

I used the DAKO Labs' developer kit (with the Biotinylated Link and Streptavidin-HRP) and DAB chromogen for the staining, and Mayer's Hemotoxylin for the counter-stain.

The results were not very good. The Pharmingen CD62L and CD3 stained sections did not show up. The GeneTex CD62L section did show up, but there appeared to be cytosolic staining (interior) instead of membrane staining (expected result).

I discussed the results with my boss, and we decided to try frozen sections on Wednesday, because the Pharmingen antibodies specified frozen sections. Originally, I was using 1/25 and 1/100 primary antibody dilutions, but this time we used 1/50 dilution. The staining results look better. There is membrane staining, but very faint.

I'd like to know if anybody has had experience using BD Bioscience Pharmingen's antibodies on PARAFFIN sections. It would be a pain to order frozen sections all the time, since they are more expensive. And my boss will need to have patients biopsied, with a good excuse, in order to get frozen sections.