Mouse anti vimentin

Serum starved and AMTN knock-down ARPE-19 cells cultured in six-well chambers or transwell inserts were washed with cold PBS, then fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 for 5 min. Next, the samples were blocked with 5% BSA for 30 min at room temperature. Cells were incubated with primary antibodies, rabbit polyclonal ZO-1 (Abcam, Cambridge, MA) diluted 1:100 in ICC buffer (1x PBS with 0.2% Tween 20, 0.5% BSA, and 0.05% sodium azide), Anti-Amelotin antibody (Catalog#ab122312, Abcam, Cambridge, MA) diluted 1: 100 in ICC buffer, anti-CRALBP (Thermo Fisher Scientific, Waltham, MA) diluted 1:500 in ICC buffer, or anti-mouse Vimentin (Santa Cruz) diluted 1:100 dilution in ICC buffer for four hours at room temperature. The antibody solution was removed, and the cells were washed with PBS. Next, samples were incubated with the anti-rabbit or anti-mouse Alexa 488 (Thermo Fisher Scientific, Waltham, MA) secondary antibody diluted 1:100 with PBS for two hours at room temperature. Slides were counter stained with DAPI (#D1306, Thermo Fisher Scientific, Waltham, MA) diluted 1:500 in PBS in the dark for one hour. After washing with 1X PBS, samples were mounted with Prolong Gold (Molecular Probes, Eugene, OR) mounting medium and examined by confocal microscopy using a Zeiss LSM 880 with Airyscan (Zeiss, USA).