Recombinant Anti-Cytokeratin 19 antibody [EP1580Y] - BSA and Azide free (ab195872)

Fluorescence multiplex immunohistochemical analysis of the human pancreas (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-Carboxypeptidase A (ab278044, magenta; Opal™690), anti-Cytokeratin 19 (ab195872, green; Opal™520) and anti-Insulin (ab181547, red; Opal™570) on human pancreas. Panel B: anti-Carboxypeptidase A stained on acinar cells. Panel C: anti-Cytokeratin 19 stained on centroacinar cells and ducts. Panel D: anti-Insulin stained on beta cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab278044 at 1/4000 dilution (0.135 μg/ml), ab195872 at 1/8000 dilution (0.127 μg/ml), and ab181547 at 1/20000 dilution (0.053 μg/ml) 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.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

Fluorescence multiplex immunohistochemical analysis of human liver tissue (formalin-fixed paraffin-embedded section). Panel A shows merged staining of ab195872 anti-Cytokeratin 19 stained on branch of bile ducts (magenta; Opal™690) at 1:8000 ( 0.127 μg/ml) [Panel B], ab275376 anti-Factor VIII stained on endothelial cells (red; Opal™570) at 1:1000 ( 0.457 μg/ml) [Panel C], and ab150301 anti-alpha smooth muscle Actin stained on smooth muscles (green; Opal™520) at 1:200 ( 0.14 μg/ml) [Panel C] on human liver. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary. 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. The section was incubated in three rounds of staining: in the order of ab195872 for 30 mins, ab275376 for 30 mins and ab150301 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used. 

Clone EP1580Y (ab195872) has been successfully conjugated by Abcam. This image was generated using Anti-Cytokeratin 19 antibody [EP1580Y] (Alexa Fluor® 647). Please refer to ab192980 for protocol details.

ab192980 staining Cytokeratin 19 in HeLa cells. The cells were fixed with 100% methanol (5 min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Triton X-100 for 1hr. The cells were then incubated with ab192980 at a working dilution of 1 in 50 (shown in red) and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1hr with an AlexaFluor^® 488 Goat anti-mouse IgG (H&L - preadsorbed) secondary (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

Image was taken with a Confocal microscope (Leica micro-systems, TCS SP8).

ab52625 staining Cytokeratin 19 in the human cell line MCF-7 (human breast carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/80. A goat anti rabbit IgG (Alexa Fluorr^® 488) at a dilution of 1/2000 was used as the secondary antibody. 

Isoytype control: Rabbit monoclonal IgG (Black) 

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue) 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625). 

Clone EP1580Y (ab195872) has been successfully conjugated by Abcam. This image was generated using Anti-Cytokeratin 19 antibody [EP1580Y] (Alexa Fluor® 488). Please refer to ab192643 for protocol details.

ab192643 staining Cytokeratin 19 in wild-type MCF7 cells (top panel) and KRT19 knockout MCF7 cells (bottom panel). The 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. The cells were then incubated with ab192643 at 1/500 dilution (shown in green) and ab195884 (Rat monoclonal to Tubulin - Alexa Fluor® 647) at 1/100 dilution (shown in red) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab52625 staining KRT19 in MCF7 cells, with negative expression in SHSY5Y 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 ab52625 at 2 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.

This data was developed using the same antibody clone in a different buffer formulation (ab52625).

This data was developed using the same antibody clone in a different buffer formulation (ab52625).

ab52625 staining Cytokeratin 19 in wild-type MCF7 cells (top panel) and KRT19 knockout MCF7 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 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. The cells were then incubated with ab52625 at 1/100 dilution 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).

Clone EP1580Y (ab195872) has been successfully conjugated by Abcam. This image was generated using Anti-Cytokeratin 19 antibody [EP1580Y] (PE). Please refer to ab224981 for protocol details. 

Overlay histogram showing HeLa cells stained with ab224981 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab224981, 1/1000 dilution) for 30 min at 22°C. 

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. 

Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter. 

Immunocytochemistry/Immunofluorescence analysis of HepG2 (human liver hepatocellular carcinoma cell line) cells labelling Cytokeratin 19 (green) with purified ab52625 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291, anti-Tubulin (mouse mAb) at 1/1000 followed by ab150120 AlexaFluor^®594 goat anti-mouse secondary (1/1000). Nuclei were counterstained with DAPI (blue).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Immunofluorescent staining of MCF-7 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab52625 at a dilution of 1/200. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200 and the cells were counter stained with DAPI.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Immunohistochemical staining of paraffin-embedded human skin with purified ab52625 at a dilution of 1/400. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Unpurified ab52625 showing positive staining in Breast carcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Unpurified ab52625 showing positive staining in Endometrial carcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Unpurified ab52625 showing negative staining in Glioma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Unpurified ab52625 showing positive staining in Gastric adenocarcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with unpurified ab52625 (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 (ab52625, 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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625). 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52625).