Immunohistochemistry was performed on normal biopsies of deparaffinized Rat cerebellum tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and 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 Rabbit Polyclonal Antibody recognizing nNOS (ab3511 ) 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 skeletal muscle tissue sections labeling nNOS (neuronal) with ab3511 at 1/400 (left). Antigen retrieval was performed using 10M sodium citrate (pH 6.0) buffer, microwaved for 8-15 minutes. Tissues were blocked in 3% BSA in PBS for 30 minutes at room temperature. A biotin-conjugated secondary antibody was used. Right - negative control.
Western blot analysis of nNOS was performed by loading 40 ?g of Mouse (Lane 1) and Rat Brain (Lane 2) tissue lysate onto a 4-12% Bis-Tris polyacrylamide gel. Proteins were transferred to a Nitrocellulose membrane. Membranes were probed with a rabbit polyclonal antibody (ab3511) recognizing nNOS at a dilution of 1:1000.
ab3511 at 1/800 dilution staining nNOS (neuronal) in rat brain tissue section by immunohistochemistry (Frozen sections).