Anti-ErbB4 / HER4 antibody [HFR-1] (ab19391)

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human Breast carcinoma 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 1:20 with a mouse monoclonal antibody recognizing ErbB4 / HER4 (ab19391) 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.

Immunocytochemistry analysis of A431 (human epidermoid carcinoma cell line) cells labeling ErbB4 / HER4 with ab19391 at 1/50 dilution (right) compared to a negative control without primary antibody (left). Cells were fixed with formalin and 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 incubated with a DyLight^® conjugated secondary antibody (green). Cells were counterstained with Anti-Actin antibody (Alexa Fluor^® 554) (red). Nuclear DNA was labelled with DAPI (blue).

Flow cytometry analysis of NIH/3T3 (mouse embryo fibroblast cell line) cells labeling ErbB4 / HER4 with ab19391 (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10⁶ cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab19391 at a dilution of 0.5 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight^® 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Heart 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 1:200 with a mouse monoclonal antibody recognizing ErbB4 (ab19391) 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.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human Pancreas 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 1:200 with a mouse monoclonal antibody recognizing ErbB4 / HER4 (ab193911) 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse placenta tissue labeling ErbB4 / HER4 with ab19391 at 1/100 dilution (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab19391 diluted in 3% BSA-PBS at a dilution of 1:100 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Immunocytochemistry analysis of NIH/3T3 (mouse embryo fibroblast cell line) cells labeling ErbB4 / HER4 with ab19391 at 1/50 dilution (right) compared to a negative control without primary antibody (left). Cells were fixed with formalin and 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 incubated with a DyLight^® conjugated secondary antibody (green). Nuclear DNA was labelled with DAPI (blue).

Immunocytochemistry analysis of SK-BR-3 (human mammary gland adenocarcinoma cell line) cells labeling ErbB4 / HER4 with ab19391 at 1/50 dilution (right) compared to a negative control without primary antibody (left). Cells were fixed with formalin and 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 incubated with a DyLight^® conjugated secondary antibody (green). Cells were counterstained with Anti-Actin antibody (Alexa Fluor^® 554) (red). Nuclear DNA was labelled with DAPI (blue).

Flow cytometry analysis of MCF7 (human breast adenocarcinoma cell line) cells labeling ErbB4 / HER4 with ab19391 (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10⁶ cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab19391 at a dilution of 1 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight^® 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Flow cytometry analysis of Hela (human epithelial cell line from cervix adenocarcinoma) cells labeling ErbB4 / HER4 with ab19391 (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10⁶ cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab19391 at a dilution of 1 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight^® 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.