Alexa fluor 594 conjugated goat anti mouse immunoglobulin g

Glass coverslips in 24-well plates were coated with 200 μl of 0.01% poly-l-ornithine solution. After aspiration and drying, 0.5 × 10⁶ cells in 500 μl of media were plated and differentiated with PMA for 18 h. Cells were washed 1× with PBS, and media were replaced with LCIS. After 20 μM nigericin stimulation for indicated time points, 500 μl 4% formaldehyde in PBS was added directly to culture medium for 2 min. After 2 min, the prefixation culture medium was replaced with 500 μl of 2% formaldehyde in PBS for 20 min. Cover slips were washed 3× with PBS and then blocked and permeabilized for 1 h in PBS containing 1% bovine serum albumin (BSA), 3% goat serum, and 0.3% Triton X-100. Coverslips were then incubated overnight at 4° overnight with primary antibody in PBS containing 1% BSA and 0.3% Triton X-100. Primary antibodies used were anti-MT-CO1, 1:1000 dilution (Invitrogen; catalog no.: 459600). Primary antibodies were visualized by staining with Alexa Fluor 594–conjugated goat antimouse immunoglobulin G (Invitrogen; catalog no.: A11005). Coverslips were counterstained and mounted with ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (Invitrogen; catalog no.: P36962) and imaged using an Olympus IX73 inverted microscope system with 4′,6-diamidino-2-phenylindole, FITC, and tetramethylrhodamine filters at 60× objective.