Immunohistochemical analysis of human paraffin-embedded cervical carcinoma tissue using ab32150 at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Lane 1: Wild-type HAP1 cell lysate
Lane 2: Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 3: Caspase-3 knockout HAP1 cell lysate
Lane 4: Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
Lanes 1 - 4: Merged signal (red and green). Green - ab32150 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32150 was shown to specifically react with pro Caspase 3 when Caspase 3 knockout samples were used. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab32150 and ab8245 (loading control to GAPDH) were diluted to 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing Jurkat cells stained with ab32150 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then 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 (ab32150, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was anti-rabbit DyLight® 488 (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (1µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.