Immunohistochemical analysis of 4% formalin fixed frozen mouse stomach tissue labeling CaSR with ab19347 at 1/100 dilution overnight at 4°C, followed by fluorophore-conjugated goat anti-mouse IgG secondary antibody for 2 hours at RT.

Fresh stomach tissue was fixed in 4% formalin for 1 hour and then incubated overnight at  in 25% sucrose before embedding in tissue freezing medium. Antigen retrieval was carried out on 8µm cryo-sections by incubating in sodium citrate buffer for 45 minutes at 4°C, immersing in sodium citrate buffer for 10 minutes at 100°C before washing 3 times for 5 minutes each in 1X PBS. Sections were then blocked in blocking buffer (0.3% Triton X-100 in 1X PBS containing 10% normal goat serum) for 30 minutes at RT before staining with ab19347.

Sections were also stained with DAPI nuclear stain (blue). Positive cells (green) were found at the base of the antral glands in the mouse stomach.

 

Immunohistochemistry was performed on normal biopsies of deparaffinized Human kidney tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing CaSR ab19347 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Overlay histogram showing SH-SY5Y cells stained with ab19347 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19347, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive result in 80% methanol (5 min) fixed SH-SY5Y cells used under the same conditions. Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

Immunohistochemistry was performed on normal biopsies of deparaffinized Human brain tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing CaSR ab19347 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

ab19347 at 1/100 staining rat brain (cerebral cortex) tissue sections by IHC-Fr. The tissue was paraformaldehyde fixed and blocked with serum before incubation with the primary antibody for 24 hours at 4°C. A biotinylated horse anti-mouse IgG was used as the secondary.

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