Mouse anti zo 1 antibody

Immunofluorescence staining of tight junction proteins was carried out to determine possible changes in cellular distribution of these proteins after 7KC treatment. Wells in duplicate were briefly washed with 1 × PBS, fixed with 4% paraformaldehyde for 15 min and washed again with 1 × PBS. Fixed cells were permeabilized with 0.3% Triton-X-100 in PBS and blocked with 6% horse or goat serum in PBS-Tx for 1 h before incubating in primary antibodies for 4 h at room temperature. Mouse anti-ZO1 antibody (1:500, Cat# ab61357, Abcam, Cambridge, UK) was used in combination with rabbit anti-claudin-5 antibody (1:100, Cat# 34–1600, Invitrogen, USA) or rabbit anti-occludin antibody (10 μg/mL, Cat# PA5-20,755, Invitrogen, USA) and stained with anti-mouse and anti-rabbit secondary antibodies conjugated with CY3 and FITC, respectively, (1:200, Thermo Fisher Scientific, USA) for 30 min with PBS washes in between and after. Immunostained coverslips were mounted on glass slides using Fluoroshield Mounting Medium with DAPI (Cat# ab104139, Abcam, UK). Images were captured with a 40X objective using excitation wavelengths of 405 nm (DAPI), 488 nm (FITC) and 543 nm (CY3) using a confocal microscope (Olympus, Tokyo, Japan).