重组Anti-Cytokeratin 14抗体[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.

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.

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.