重组Anti-Cytokeratin 18抗体[E431-1] (ab32118)

Flow cytometry overlay histogram showing left MCF7 positive cells and right negative A375 stained with ab32118 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab32118) (1x 10⁶ in 100μl at 0.008μg/ml (1/258750)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in MCF7 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

 

All lanes : Anti-Cytokeratin 18 antibody [E431-1] (ab32118) at 1/2000 dilution (Purified)

Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 48 kDa
Observed band size: 48 kDa

Immunocytochemistry/Immunofluorescence analysis of HeLa (+ve) and U87-MG (-ve) cells labelling Cytokeratin 18 with ab32118 at 2 ug/ml overnight at +4°C. Cells were fixed with 100% Methanol (5 min), 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. A preadsorbed Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150081) at 1/1000 dilution was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000), using ab150119, a preadsorbed  Alexa Fluor® 647-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 4% formaldehyde (10 min).

Immunocytochemistry/Immunofluorescence analysis of HT-29 cells labelling Cytokeratin 18 with purified ab32118 at 1/500. Cells were fixed with 100% Methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used followed by anti-mouse secondary antibody (ab150120).
For negative control 2, mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) were used.

Alexa Fluor^® 488 (ab194124) conjugated version is available for this clone.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human stomach tissue sections labeling Cytokeratin 18 with purified ab32118 at 1/500 dilution (0.08 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Cytokeratin 18 with purified ab32118 at 1/20 dilution (2µg/ml) (red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

R-PE (ab210410) conjugated version is available for this clone. 

 

 

Indirect immunofluorescence staining with antibodies against DNA, cytokeratin-7 and cytokeratin-18 (upper panel) or against DNA, vimentin and cytokeratin-18 (lower panel). Representatives are presented. Scale bar: 20 µm.

Control or treated cells were fixed for 15 min with 4% PFA containing 0.1% Triton X-100 at room temperature. The following primary antibodies were used for staining: monoclonal mouse antibodies against vimentin and cytokeratin-7 (both 1:100, DAKO) and monoclonal rabbit antibody against cytokeratin-18 (1:50, Abcam). DNA was stained using DAPI (4’,6-diamidino-2-phenylindole-dihydrochlorid) (Roche). Slides were examined using an Axio Imager 7.1 microscope (Zeiss) and images were taken using an Axio Cam MRm camera (Zeiss). The immunofluorescence stained slides were also examined by a confocal laser scanning microscope (CLSM) (Leica CTR 6500, Heidelberg). Images were processed using Photoshop.

ab32118 showing positive staining in Normal colon tissue.

Anti-Cytokeratin 18 antibody [E431-1] (ab32118) at 1/10000 dilution + A431 cell lysate

Predicted band size: 48 kDa
Observed band size: 48 kDa

Overlay histogram showing MCF7 cells stained with ab32118 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab32118, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF7 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

 

ab32118 showing positive staining in Gastric adenocarcinoma tissue.

Fluorescent immunohistochemical analysis of paraffin-embedded human normal kidney tissue using ab32118. Green-CK18 red-PI

ab32118 showing positive staining in Normal kidney tissue.

ab32118 showing positive staining in Breast carcinoma tissue.

ab32118 showing negative staining in Glioma tissue.

ab32118 showing negative staining in Normal brain tissue.