Anti casr antibody

Western blot was performed using a standard protocol. Human MCs were starved for 24 h in a serum-free medium prior to stimulation with high [Ca²⁺]_o. At the end of the 24 h incubation, the cells were harvested for western blot analysis. Anti-CaSR antibody (Affinity BioReagents, USA), anti-TRPC1, -TRPC3, -TRPC4, or -TRPC6 antibodies (Alomone Labs, Israel) or an anti-actin antibody (Santa Cruz, USA) were used as primary antibodies. Fluorescence-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies (Invitrogen, USA) were used as secondary antibodies. Western blot bands were quantified using the Odyssey infrared imaging system (LI-COR Bioscience, USA).

Immunofluorescence staining was performed on cultured MCs growing on coverslips using a standard protocol. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.4% Triton X-100 in PBS for 60 min at room temperature. The nonspecific binding sites were blocked with 50% goat serum in PBS for 60 min at 37°C. The cells were then incubated overnight at 4°C with anti-CaSR antibody (Affinity BioReagents, USA). After washing, cells were stained with Alexa Fluor 594 conjugated to goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR) for 60 min at room temperature. Secondary antibody without prior antibody treatment was also included as negative controls. Cells were then stained with DAPI (Sigma, USA) for 15 min at room temperature to detect nuclei. After washing, samples were examined under a laser scanning confocal microscope (FV300; Olympus, Japan). Calibrations were performed immediately following each experiment. More than 50 cells were inspected per experiment, and photos of cells with typical morphology and staining are presented.