Anti-Cytokeratin 14 antibody [SP53]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

ab119695 staining KRT14 in wild-type A431 cells (top panel) and KRT14 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab119695 at 5μg/ml concentration 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 red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

Flow cytometry analysis of A431 (human epidermoid carcinoma) labeling Cytokeratin 14 with purified ab119695 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as a secondary antibody. Isotype control -Rabbit monoclonal IgG (ab172730) (Black). Unlableled control -Unlabelled cells (blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

Human prostate tissue stained with ab119695 at a dilution of 1/100.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

This data was developed using the same antibody clone in a different buffer formulation (ab236439). Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast labelling Cytokeratin 18 with ab93741 at 1/200 dilution (1.02 µg/mL) (B), Collagen VI with ab271938 at 1/500 dilution (2.084 μg/ml) (C) and Cytokeratin 14 with ab236439 at 1/2000 dilution ( 0.519 μg/ml) (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. Panel A : merged staining of anti-Cytokeratin 14 (magenta; Opal™690), anti-Cytokeratin 18 (green; Opal™520) and anti-Collagen VI (red; Opal™570) on human breast. Panel B : anti-Cytokeratin 18 stained on luminal epithelial cells. Panel C : anti-Collagen VI stained on stroma. Panel D : anti-Cytokeratin 14 stained on myoepithelial cells. The section was incubated in three rounds of staining : in the order of ab236439 for 30 mins, ab93741 for 10 mins, and ab271938 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

Immunocytochemistry/ Immunofluorescence analysis of A431(human epidermoid carcinoma epithelial cell) cells labeling Cytokeratin 14 with purified ab119695. 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)). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

Immunohistochemistry (Frozen) analysis of rat skin tissue section labeling Cytokeratin 14 with purified ab119695. Sections were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Antigen retrieval was heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

Immunohistochemistry (Frozen) analysis of mouse skin tissue section labeling Cytokeratin 14 with purified ab119695. Sections were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Antigen retrieval was heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Lab

Western blot - Anti-Cytokeratin 14 antibody [SP53] (AB119695)

Lanes 1 - 4 : Merged signal (red and green). Green - ab119695 observed at 52 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab119695 was shown to react with KRT14 in A431 wild-type cells in Western blot. Loss of signal was observed when KRT14 knockout sample was used. A431 wild-type and KRT14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab119695 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 93 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cytokeratin 14 antibody [SP53] (ab119695) at 1/93 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human KRT14 (Cytokeratin 14) knockout A-431 cell lysate (<a href='/en-us/products/cell-lysates/human-krt14-cytokeratin-14-knockout-a-431-cell-lysate-ab261706'>ab261706</a>) at 20 µg

Lane 3:

Human skin whole tissue lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa,37 kDa

false