Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

ab181595 staining Cytokeratin 14 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 ab181595 at 0.2μg/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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Immunohistochemical analysis of paraffin-embedded human skin tissue labeling Cytokeratin 14 with ab181595 at 1/2000 dilution, followed by prediluted HRP polymer for Rabbit/Mouse IgG. Basal cells of epidermis show strong staining while no staining on the stratum corneum. Counterstained with hematoxylin.

Negative control : PBS instead of primary antibody, secondary antibody is prediluted HRP polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of cervix tissue labeling Cytokeratin 14 with ab181595 at 1/2000 dilution, followed by prediluted HRP polymer for Rabbit/Mouse IgG. Squamous carcinoma cells show strong staining. Counterstained with hematoxylin.

Negative control : PBS instead of primary antibody, secondary antibody is prediluted HRP polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Negative control image : IHC image of Cytokeratin 14 staining in a section of formalin-fixed paraffin-embedded A431 KO cell pellet block performed on a Leica Biosystems BOND^® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab181595, 0.01ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Lab

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

IHC image of Cytokeratin 14 staining in a section of formalin-fixed paraffin-embedded A431 WT cell pellet block performed on a Leica Biosystems BOND^® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab181595, 0.01ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Lab

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Immunohistochemical analysis of paraffin-embedded mouse skin tissue labeling Cytokeratin 14 with ab181595 at 1/2000 dilution, followed by prediluted HRP polymer for Rabbit/Mouse IgG. Basal cells of epidermis show strong staining while no staining on the stratum corneum. Counterstained with hematoxylin.

Negative control : PBS instead of primary antibody, secondary antibody is prediluted HRP polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Immunofluorescent analysis of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized PC-12 (Rat adrenal gland pheochromocytoma cells) labeling Cytokeratin 14 with ab181595 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm staining on PC-12 cell line is observed. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls : -
-ve control 1 : ab181595 at 1/1000 dilution followed by ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor^®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Intracellular Flow Cytometry analysis of PC-12 (rat adrenal gland pheochromocytoma) cells labeling Cytokeratin 14 with purified ab181595 at 1/190 dilution (10μg/mL) (red).

Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Immunohistochemical analysis of paraffin-embedded rat skin tissue labeling Cytokeratin 14 with ab181595 at 1/2000 dilution, followed by prediluted HRP polymer for Rabbit/Mouse IgG. Basal cells of epidermis show strong staining while no staining on the stratum corneum. Counterstained with hematoxylin.

Negative control : PBS instead of primary antibody, secondary antibody is prediluted HRP polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Blocking/Dilution buffer : 5% NFDM/TBST.

HaCaT cells express a higher level of Cytokeratin 14 than other cell lines such as A431.

All lanes:

Western blot - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (ab181595) at 1/20000 dilution

Lane 1:

A431 (Human epidermoid carcinoma) whole cell lysate at 20 µg

Lane 2:

HaCaT (Human keratinocyte cells) whole cell lysate at 20 µg

Lane 3:

Mouse skin lysate at 20 µg

Lane 4:

Rat skin lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 53 kDa

false

• WB

Supplier Data

Western blot - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (AB181595)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (ab181595) at 1/20000 dilution

All lanes:

Human skin lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 53 kDa

false