Rabbit anti orai1 antibody

Isolated osteoblasts were plated in a 24 well plate at a density of 1×10⁴/cm² and incubated with 10%FBS/αMEM for 24 hours. Upon assay, cells were treated with 150 nM of thapsigargin (Enzo Life Sciences, Inc.) for 5 minutes at RT to deplete ER calcium stores, and immediately fixed with 4% paraformaldehyde for 10 minutes. After several rinses with PBS, cells were permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 10% goat serum for 30 minutes. Cells were then incubated with rabbit anti-stim1 antibody (MyBioSource; 1:100 dilution) for overnight at 4°C followed with the secondary antibody DyLight 488 anti-rabbit IgG conjugate (Vector Labs, 1:300 dilution) for 1 hour at RT. After several rinses with 1X PBST containing 0.05% Tween 20, cells were incubated with rabbit anti-orai1 antibody (Alomone Labs, 1:66 dilution) at 4°C overnight followed with the secondary antibody Cy3-anti-rabbit IgG conjugate (Millipore, 1:100 dilution). Fluorescence was visualized under a ZEISS fluorescence microscope (Axio Observer) equipped with the ZEN 3.0 (blue edition) software. Auto-exposure was selected for all selected images. Cell number was counted with the Image J software program.