Showing representative confocal laser scanning slice images for tight junction proteins. Stimulation with anti-CD24 peptide antibody led to increases in tight junction proteins and altered cellular localization patterns indicative of functional tight junction complexes. Cells were grown on permeable Transwell filters until confluence as described in Methods. Data are representative of three independent experiments. (A) Phase contrast images showing confluence of cultures (cell density: mean value 3.2 × 104/cm2) at 3 h after challenged with isotype control antibody (left) and with CD24 peptide antibody (right) prior to fixation. (B) Left panel shows confocal optical sections of representative monolayers that were challenged with isotype control antibody for 3 h before processing: (a) staining with mouse isotype control antibody; (b) tight junction protein marker ZO-1 showing focal staining principally at cell borders; (c) tight junction protein marker ZO-2 showing scattered cytoplasmic reaction; (d) occludin showing very weak reaction; (e) claudin-7 showing faint peripheral location. Right panel shows confocal optical sections of representative monolayer cultures that were challenged with anti-CD24 peptide antibody for 3 h before processing: (f) negative control show trace non-specific staining with mouse isotype control antibody; (g) ZO-1 well formed and localized at cell-cell contact; (h) ZO-2 expressed in 60 percent of the area of an optical section; (i) occludin showing peripheral distribution compared to trace cytoplasmic staining in monolayers exposed to control antibody (see d); (j) claudin-7 showing strong reaction that is diffuse near the cell periphery. This optical section image was captured at a deeper level. Claudin 3 and par-6 were not detected in isotype control or anti-CD24 antibody treated cultures. Bars, 20 μm. Note that the optical images were captured at a level corresponding to maximum staining in each case.