Formalin-fixed, paraffin-embedded human colon adenocarcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and TP53 knockout HAP1 cells stained with ab16665 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1%PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab16665) (1x10⁶ in 100 µl at 0.008 µg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line TP53 knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody can also be used in HAP1 cells fixed with 80% methanol (5 min) / permeabilized with 0.1%PBS-Triton X-100 for 15 min used under the same conditions.

Flow cytometry analysis of MCF7 (human breast adenocarcinoma epithelial cell) labeling p53 with purified ab16665 at 1/200 dilution (0.81 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730)(black). Unlableled control - Unlabelled cells (blue).

ab16665 staining p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16665 at 1/25 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling p53 with purified ab16665 at 1/25 (6.5 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Formalin-fixed, paraffin-embedded human lung adenocarcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human stomach adenocarcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human ovarian adenocarcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human bladder transitional cell carcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human hepatocellular cell carcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human cervical squamous cell carcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast ductal cell carcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human B cell lymphoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.