Anti-p63 antibody [EPR5701] - BSA and Azide free

• IHC

Lab

Immunohistochemistry - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

Immunohistochemical analysis of formalin fixed paraffin embedded human prostate labelling p63 with ab124762 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab124762 anti-p63 [EPR5701] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling p63 with Purified ab124762 at 1/80 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Immunocytochemistry/ Immunofluorescence analysis of A431(Human epidermoid carcinoma epithelial cell) cells labeling p63 with Purified ab124762 at 1/200 dilution (4 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast tissue sections labeling p63 with Purified ab124762 at 1/5000 dilution (0.16 μg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Unpurified ab124762 staining p63 in Human corneal limbal epithelial cells (primary culture) by ICC/IF (immunocytochemistry/immunofluorescence). Cells were fixed with methanol and permeabilized with 0.3% Triton X-100 for 5 minutes. Samples were incubated with primary antibody (1/100 in PBS + 10% Goat serum) for 18 hours at 4°C. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

This image is courtesy of an Abreview submitted by Manuel Chacon

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Clone EPR5701 (ab214790) has been successfully conjugated by Abcam. This image was generated using Anti-p63 antibody [EPR5701] (Alexa Fluor® 488). Please refer to ab246727 for protocol details.

ab246727 staining p63 in A431 cells. The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab246727 at 1/100 dilution (shown in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in A431 cells fixed with 4% formaldehyde (10 min).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Clone EPR5701 (ab214790) has been successfully conjugated by Abcam. This image was generated using Anti-p63 antibody [EPR5701] (Alexa Fluor^® 647). Please refer to ab246728 for protocol details.

Overlay histogram showing A431 cells stained with ab246728 (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 / 10% normal Goat serum to block non-specific protein-protein interactions followed by the antibody (ab246728) (1x 10⁶ cells in 100μl at 0.08μg/ml (1/6250 dilution)) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter.

This antibody gave a positive signal in A431 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

Fluorescence multiplex immunohistochemical analysis of human prostate gland tissue (formalin/PFA-fixed paraffin-embedded section). Panel A : merged staining of anti-p63 (ab124762, magenta; Opal™690), anti-Cytokeratin 5 (ab236216, green; Opal™520) and anti-Prostate Specific Antigen (ab76113, red; Opal™570) on human prostate gland tissue. Panel B : anti-Prostate Specific Antigen stained on luminal cells. Panel C : anti-Cytokeratin 5 stained on cytoplasm of basal cells. Panel D : anti-p63 stained on nucleus of basal cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining : in the order of ab124762 (1/5000), ab236216 (1/400), and ab76113 (1/2000) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

• WB

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Western blot - Anti-p63 antibody [EPR5701] - BSA and Azide free (AB214790)

The bands observed are consistent with what have been described in PMID 30649915 as isoforms of p63.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124762).

All lanes:

Western blot - Anti-p63 antibody [EPR5701] (<a href='/en-us/products/primary-antibodies/p63-antibody-epr5701-ab124762'>ab124762</a>) at 1/1000 dilution

Lane 1:

HaCaT (Human skin keratinocyte) whole cell lysates at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

Mouse skin lysates at 20 µg

Lane 4:

Mouse thymus lysates at 20 µg

Lane 5:

Mouse bladder lysates at 20 µg

Lane 6:

Rat skin lysates at 20 µg

Lane 7:

Rat bladder lysates at 20 µg

Lane 8:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 77 kDa

Observed band size: 37-75 kDa

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