Recombinant Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (ab76126)

False colour image of Western blot: Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab76126 was shown to bind specifically to Cytokeratin 20. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

ab76126 staining Cytokeratin 20 in HT-29 cells, with negative expression in MCF7 cells. The cells were fixed with 100% methanol (5 min), permeabilised 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 ab76126 at 0.1 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 µg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

This product also work with 4% formaldehyde (10 min) fixation under the same testing conditions.

ab76126 at 1/100 dilution staining Cytokeratin 20 in human colon adenocarcinoma by Immunohistochemistry, Paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HT-29 (human colorectal adenocarcinoma) cells labelling Cytokeratin 20 with purified ab76126 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

Primary: ab76126, 1/50000 dilution
Sample: human small intestine lysate, 10 ug
Secondary: goat anti-rabbit-HRP, 1/1000 dilution

Overlay histogram showing LoVo (Human colorectal adenocarcinoma cell line) cells stained with ab76126 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76126, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. 

Primary: ab76126, 1/50000 dilution
Sample: ab73640, 0.01 ug
Secondary: ab97080, 1/5000 dilution

ab76126 showing positive staining in human colonic adenocarcinoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab76126 showing positive staining in human urinary bladder transitional carcinoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab76126 showing negative staining in human hepatocellular carcinoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab76126 showing negative staining in human breast carcinoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Fluorescent immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue using ab76126. Green-CK20 red-PI

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.