Fig. 2. Extractability of CEACAM1-L and CEACAM1-S. (A) Cells were grown on Petri dishes. Fixation was performed with 3% paraformaldehyde for 30 minutes. Upper panel: the cells were extracted with the indicated concentrations of Triton X-100 for 10 minutes at room temperature, and the soluble (S) and insoluble (I) fractions were analyzed by immunoblotting. Lower panel: fixed cells were extracted with absolute methanol for 2 minutes at room temperature or for 6 minutes at -20°C, and the soluble (S) and insoluble (I) fractions were analyzed by immunoblotting. (B) CEACAM1-L-transfected cells were grown on permeable filters, fixed with 3% paraformaldehyde for 30 minutes, stained by CC16 after the indicated permeabilization procedure and viewed by confocal microscopy. (a) Permeabilization with 0.05% Triton X-100 for 10 minutes. (b) Permeabilization with 0.1% Triton X-100 for 10 minutes. (c) Permeabilization with methanol for 6 minutes at -20°C. (d) Permeabilization with methanol for 2 minutes at room temperature. Permeabilization with methanol for 2 minutes at room temperature gave the best preservation of the lateral staining of CEACAM1-L. The xy-fields represent the image in one focal plane (the xy-dimension) parallel to the basal surfaces (the filter plane) of the cells. The images labelled z represent the third, vertical z-dimension, obtained by computer reconstruction of all the focal planes analyzed in the xy-dimension. The lines in the xy-fields indicate the vertical planes that are shown in the z-reconstructions. The lines in the z-reconstructions indicate the level of the focal planes shown in the xy-fields.