Mice received i.p. injections of 5 IU eCG. Forty-eight hours later, the mice received injections via the tail vein of 250-300 (xg of the following proteins in 150-|xl total volumes: cationized lal, rabbit IgG, anionized rabbit IgG, BSA, human a2-M, or cationized a2-M. Permeability of the blood-follicle barrier increases with the administration of anesthesia because of its secondary effects of increasing blood flow and smooth muscle relaxation (data not shown). Therefore, tail vein, rather than carotid, injections were performed so that anesthesia would be unnecessary. Immediately after tail vein injections, those mice that had received injections of anionized rabbit IgG and human a2-M received i.p. injections of 5 IU hCG. Three hours after protein injection, the mouse ovaries were collected, fixed in fresh 4% paraformaldehyde, sectioned, and used for immunocy-tochemistry. buy ortho tri-cyclen
Fixed ovaries were sectioned (8 |лт) using a cryotome (Leitz, Wetzlar, Germany) and incubated with primary antibodies (1:1000, rabbit anti-BSA, rabbit anti-human Ial, or rabbit anti-human a2-M) overnight at 4°C. Slides containing 15-20 ovary sections were washed, incubated with secondary antibody (1:1000 FITC-goat anti-rabbit IgG), and observed with a Nikon fluorescence microscope (Nikon Corp., Melville, NY) as previously described.