Sc 33735

Cells were grown on glass coverslips and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Cells were blocked in 5% BSA for 1hr at room temperature. Cells were stained with the PMA1 antibody (1:100; SC-33735, Santa Cruz Biotechnology) or CA-IX antibody for 2 hours (1:500, ab184630, Abcam) and washed in PBS. Cells were further incubated for 1 h with secondary anti-rabbit-Alexa Fluor 594 antibody (1:2000; A11072, Invitrogen) or anti-mouse-Alexa Fluor 594(1:2000; A11005, Invitrogen) and additionally incubated in WGA, cell membrane marker (W6748, Invitrogen) for 10 min on ice. The cells were mounted for fluorescence with DAPI (H-1200, Vector). The slides were viewed by Leica inverted SP5 AOBS confocal microscope, and micrographs were taken, and images were subsequently acquired in the Moffitt Analytic Microscopy Core Facility by using dual photomultiplier tube detectors and LAS AF software (Leica Microsystems). For detection of intracellular PMA1, cells were fixed and permeabilized with 1:1 mixture of methanol and acetone, and immunostained with PMA1 antibody for 1h, followed by 1 hour of incubation with the secondary anti-rabbit Alexa488 antibody (Molecular Probes, Invitrogen). The cells were mounted and viewed by fluorescence microscopy.