Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity

The procedures for RIA, immunofluorescence on semi-thin frozen sections, and electron microscopy have been described in detail elsewhere (61, 63, 82, 83) . Briefly, MDCK cells were plated at immediate confluency on 50well detachable tissue culture dishes (,',,100,000 cells per well; Lux, Miles Laboratories, Inc., Naperville, IL) and fixed 24 h later with either 2 % formaldehyde (freshly prepared from paraformaldehyde [PFAI) at room temperature, or 96% methanol at -20°C. The monolayers were then sequentially treated with 50 mM NH4CI in PBS, 1% BSA (Sigma) in PBS supplemented with 50 o.g/ml preimmune goat IgG, mAb, and affinity-purified ~25I-goat anti-mouse IgG. The wells were washed, dried, detached, and counted in a gamma counter (Hewlett-Packard Co., Palo Alto, CA).