Immunofluorescent Labeling of Tight and Adherens Junctions

Cell monolayers cultured on transwell inserts were fixed in cold methanol. For immunofluorescent labeling of TJ and AJ proteins, cell monolayers were incubated with the antibodies mentioned above. Briefly, membranes were blocked with 5% bovine serum albumin in PBS, followed by primary antibody incubation. Membranes were then rinsed three times in PBS, followed by secondary antibody incubation. Furthermore, membranes were rinsed three times in PBS, stained with DAPI, and mounted with an anti-fading mounting medium (Vector Laboratories, Cat. No, H-1000, Burlingame, CA) (26 (link), 30 (link), 31 (link)). Samples were imaged with ×63 objectives using a Leica TCS-SP8-AOBS inverted confocal microscope (Leica Microsystems, Wetzlar, Germany). Images were processed with Adobe Photoshop (RRID:SCR_014199) software.