I have been trying to optimize CD31 in frozen rat brain using Pharmingen's
clone TLD-3A12 for rat CD-31. The rats have gloisarcoma tumors and I want to
look at tumor vascularity. I can get great, clean staining in the
non-tumorous areas of the brain, but the tumors are a mess of nonspecific
staining where all cells are picking up the chromogen and nothing specific
can be discerned. The method is acetone fixation, ABC with DAB/NiCl. H2O2
blocking, serum blocking, avidin/biotin blocking, non-serum blockers and
biotin-free detection have all been tried as have zinc/tris and formalin
fixation. Negative controls are clean. I' m in the process of looking at the
gliosarcoma cell line to see if  the antibody recognizes the tumor cells in
vitro.  Just wondering if anyone has had a similar experience??

I too like Fawlty Towers, but I also enjoy 'Keeping Up Appearances'.

Brett     

Brett M. Connolly, Ph.D.
Merck Research Laboratories
Dept. of Pharmacology
WP26A-3000
PO Box 4
West Point, PA 19486
Ph. 215-652-2501
fax. 215-652-2075
e-mail. brett_connolly@merck.com