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 - ab32499 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32499 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. ab32499 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab32499).

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma ab32499 at 1/250 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab32499)

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

ICC/IF image of ab32499 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 4% formaldehyde (10 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32499, 5 µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, anti-rabbit DyLight^® 488 used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab32499)

Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with ab32499 (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 followed by the antibody (ab32499, 1/50 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 (monoclonal) (1 µg/1 x 10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab32499)