ab5586 labelling nNOS (neuronal) in the cytoplasm of Mouse heart tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method 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. Tissue sections were incubated with the primary antibody (1:20 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit IgG was used as the 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/Immunofluorescence analysis of nNOS (neuronal) (green) showing staining in the cytoplasm and membrane of U251 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 incubated with ab5586 in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC 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.

ab5586 labelling nNOS (neuronal) in the cytoplasm of Human testis tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method 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. Tissue sections were incubated with the primary antibody (1:200 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit IgG was used as the 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.

ab5586 labelling nNOS (neuronal) in the cytoplasm of Human skeletal muscle (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method 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. Tissue sections were incubated with the primary antibody (1:200 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit IgG was used as the 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.