Immunofluorescent analysis of Calcium Sensing Receptor (green) showing staining in the cytoplasm and nucleus of PC12 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Calcium Sensing Receptor polyclonal antibody (Product # PA1-934A) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Western blot analysis of Calcium Sensing Receptor was performed by loading 25 µg of U251 (lane 1) and rat brain (lane 2) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a Calcium Sensing Receptor polyclonal antibody (Product # PA1-934A) at a dilution of 1:500 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~120 kDa.