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.

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).

All lanes : Anti-Cytokeratin 14 antibody [SP53] (ab119695) at 1/93 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : KRT14 knockout A431 whole cell lysate
Lane 3 : Human skin whole tissue lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 52 kDa

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.

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.

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).

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.

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