Fig. 8. Double-staining for CEACAM1-L with monoclonal and polyclonal anti-CEACAM1 antibodies. Confluent, polarized CEACAM1-L-expressing cells were first stained with mAb 5.4/Alexa488-conjugated secondary antibodies (green colour), then with CC16/Alexa546-conjugated secondary antibodies (red colour). The yellow colour indicates apparent colocalization. (A) Non-permeabilized cells, which were treated with or without hyperosmotic sucrose, were analyzed at standard resolution (voxel: 0.07x0.07x0.35 µm³). A composite of all the focal planes for the entire apical region above the tight junction is shown for each sample. (B) Non-permeabilized cells, treated with hyperosmotic sucrose. Several focal planes (0.2 µm) were scanned through the apically located microvillar region, at two different xy-resolutions. In the upper part of the figure the higher resolution corresponds to a voxel size of 0.04x0.04x0.2 µm³; in the lower part of the figure the same cells were scanned at lower resolution corresponding to a voxel size of 0.07x0.07x0.2 µm³. The z-reconstructions through the microvillar region are from the same z-plane, as indicated by the horisontal lines in the xy-panels. Note that the apparent colocalization of the different epitopes at the lower resolution disappeared at the higher resolution. (C) Permeabilized cells treated with or without hyperosmotic sucrose. Voxel size, 0.04x0.04x0.2 µm³. The upper panels (marked `Above tight junction') represent an xy-plane through the apical portion of two adjacent cells. The middle panels (marked `Below tight junction') represent an xy-plane through the middle portion of the same cells, half-way between the apical and the basal surfaces. The lower panels (marked z) represent a z-reconstruction through the border between the adjacent cells at the location indicated by the arrows in the upper and middle panels. Note that in the non-treated cells only the polyclonal epitopes (O-epitopes) were exposed, whereas in cells treated with hyperosmotic sucrose the monoclonal epitope (N-epitope) became exposed in the area below the tight junctions. (D) A model of apical microvilli analyzed at two pixel sizes. The microvilli of the apical surfaces in kidney tubular cells are uniform in size and organized in a hexagonal pattern. Here we show an xy-plane through the apical region, where we have drawn the microvilli, the intermicrovillar distances and CEACAM1 to scale. The average diameter of brush border microvilli is 150 nm, the smallest intermicrovillar distance is around 30 nm and the length of the 4 Ig domain extracellular domain of CEACAM1 is approximately 15 nm. The hexagonal arrangement of the microvilli allow for antiparallel CEACAM1-CEACAM1 binding at the narrowest inter-microvillar distances, while at other locations CEACAM1 would not be engaged in antiparallel binding. In the upper part of the figure we put a raster on this model with squares corresponding to 0.04x0.04 µm², in the lower part of the figure we put a raster on the model with squares corresponding to 0.07x0.07 µm², that is, the dimensions of the pixels in Fig. 8B. Squares (pixels) that contained only antiparallel, bound CEACAM1 (corresponding to the state with masked N-epitope and exposed O-epitopes) were given a red color, squares (pixels) that contained only CEACAM1 not engaged in antiparallel binding (corresponding to the state with exposed N-epitope and masked O-epitopes) were given a green color and squares (pixels) that contained both states of CEACAM1 were given a yellow color. This showed that with a pixel size of 0.07x0.07 µm², the majority of the pixels became yellow, whereas the pixels of 0.04x0.04 µm² became almost exclusively red or green. See the Discussion for further details.