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Afterthree washes in TBS with 0.1% Tween 20 and 15-min block in TBS with milk, membranes were incubated with a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After five further washes, antibody binding was detected with the Western Lightning Chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA) and Kodak X-Omat blue XB-1 films. Immunogold Staining for Electron Microscopy Rats were perfused as described above with a solution containing 4% PLP and 0.01% glutaraldehyde in PBS. Small pieces of tissue were cryoprotected in 2.3 M sucrose in PBS. Ultrathin cryosections were cut on a Leica EM FCS at —80°C and collected onto formvar-coated gilded 200-mesh grids. Sections were blocked for 10 min on drops of 5% (v/v) normal goat serum plus 1% (w/v) bovine serum albumin in PBS. The anti-subunit A antibody was applied at a final concentration of 1:100 in DAKO diluent (DAKO Corp., Carpinteria, CA) for 1 h at room temperature. Following several washes in PBS, the antibody was labeled with goat anti-rabbit IgG conjugated to 15-nm gold particles (Ted Pella, Inc, Redding, CA) at a final concentration of 1:20 in DAKO diluent for 1 h at room temperature. After several rinses in distilled water, the sections were stained on ice-cold drops of uranyl acetate/tylose solution for 10 min and allowed to air-dry. The sections were examined in a Philips CM 10 transmission electron microscope at 80 kV.