A novel modification of the avidin-biotin complex method for immunohistochemical studies of transgenic mice with murine monoclonal antibodies.
When mouse tissues are probed with murine monoclonal antibodies (MAb) by indirect immunohistochemistry, the secondary antibody detects tissue-bound MAb and irrelevant, endogenous mouse immunoglobulins. The latter are a source of confounding background, especially in diseased tissues. To circumvent this problem, we generated complexes of primary MAb and biotinylated secondary antibodies in vitro for use as antigen-specific probes. After blocking free binding sites in the complexed secondary antibodies with normal mouse serum, the complexes were applied to mouse tissue sections and tissue-bound complexes were visualized with an avidin-biotin detection system. Complexes formed with 12 different rat or mouse MAb were used to probe sections of normal mice, tumor-bearing transgenic mice, and mice with tumor xenografts. The staining patterns produced by these probes reflected the specificity of the MAb in the complexes, and the labeling of irrelevant, endogenous mouse immunoglobulins was reduced substantially. This novel, indirect immunohistochemical method can be exploited to study normal and diseased mouse tissues using a variety of murine MAb.
Fung
K M
KM
Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Messing
A
A
Lee
V M
VM
Trojanowski
J Q
JQ
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D002531:Cerebellum / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008822:Mice, Transgenic; D008875:Middle Aged; D009368:Neoplasm Transplantation
D000911:Antibodies, Monoclonal; D001360:Avidin; D001710:Biotin