Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human heart tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10 mM sodium citrate (pH 6.0) 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 1:20 with a rat monoclonal antibody recognizing HCN4 ab32675 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.

Anti-HCN4 antibody [SHG 1E5] (ab32675) at 1/200 dilution + Rat eye lysate at 25 µg

Secondary
HRP Conjugate

Developed using the ECL technique.

Predicted band size: 130 kDa
Observed band size: 132 kDa why is the actual band size different from the predicted?

Overlay histogram showing PC-12 (Rat adrenal gland pheochromocytoma cell line) cells stained with ab32675 (red line). The cells were fixed with methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32675, 1/50 dilution) for 30 minutes at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rat IgG1 [RTK2071] (ab18412, 2 µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC-12 cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized with 0.1% PBS-Tween used under the same conditions.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human tonsil tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10 mM sodium citrate (pH 6.0) 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 1:20 with a rat monoclonal antibody recognizing HCN4 ab32675 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.