Fig. 3. Confocal microscopy of polarized CEACAM1-L and CEACAM1-S expressing MDCK cells. CEACAM1-L- (a,c,e,g,i,k) and CEACAM1-S- (b,d,f,h,j,l) expressing cells were immunostained with anti-CEACAM1 CC16 antibodies (a-j) or pre-immune immunoglobulins (k,l) and FITC-labelled secondary antibodies. The cells were either permeabilized (a-f) by methanol for 2 minutes at room temperature or unpermeabilized (g-l) before staining. (a) is a z-reconstruction of the cells shown in xy-projections in (c) and (e), (b) is a z-reconstruction of the cells shown in xy-projections in (d) and (f), (g) is a z-reconstruction of the cells shown in xy-projection in (i), and (h) is a z-reconstruction of the cells shown in xy-projection in (j), respectively. The focal planes of (c,d,i,j,k,l) run through the apical cell surfaces. The focal planes of (e) and (f) run in the middle, between the apical and the basal cell surfaces. Apical staining for CEACAM1-L and CEACAM1-S is seen under all conditions, but lateral staining is only seen for CEACAM1-L and under permeabilizing conditions (a,e). Note that there is no lateral staining for CEACAM1-S (b and f). Bars: 20 µm.