Recombinant Anti-Cytokeratin 19 antibody [EP1580Y] - Cytoskeleton Marker (ab52625)

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.

This data was developed using ab195872, the same antibody clone in a different buffer formulation. 

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. 

This data was developed using ab195872, the same antibody clone in a different buffer formulation. 

10-color fluorescence multiplex immunohistochemical analysis of human lung cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-FOXP3 (ab215206; Cyan; TG540N), anti-PD1 (ab52587; Red; TG700N), anti-CD163 (ab182422; Brown; TG650N), anti-HLA-DR (ab92511; Yellow; TG570N), anti-CD4 (ab133616; Violet; TG620N), anti-CD8 alpha (ab101500; Purple; TG540S), anti-CD20 (ab9475; Grey; TG660S), anti-CD68 (ab192847; Green; TG520N), anti-Cytokeratin 19 (ab52625; Light blue; TG440N). TG470SN (dark blue) was used as a nuclear counter stain. The inset image shows the separate Cytokeratin 19 signal.

The section was incubated in nine rounds of staining; in the order of ab215206 (1/100 dilution), ab52587 (1/200 dilution), ab182422 (1/300 dilution), ab92511 (1/200 dilution), ab133616 (1/600 dilution), ab101500 (1/300 dilution), ab9475 (1/100 dilution), ab192847 (1/300 dilution), ab52625 (1/400 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

Image acquisition was performed with TissueFAXS Spectra (TissueGnostics).

10-color fluorescence multiplex immunohistochemical analysis of human lung cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-FOXP3 (ab215206; Cyan; TG540N), anti-PD1 (ab52587; Violet; TG700N), anti-CD163 (ab182422; Red; TG650N), anti-HLA-DR (ab92511; Yellow; TG570N), anti-CD4 (ab133616; Orange; TG620N), anti-CD8 alpha (ab101500; Purple; TG540S), anti-CD20 (ab9475; Grey; TG660S), anti-CD68 (ab192847; Green; TG520N), anti-Cytokeratin 19 (ab52625; Light blue; TG440N). TG470SN (dark blue) was used as a nuclear counter stain.

The section was incubated in nine rounds of staining; in the order of ab215206 (1/100 dilution), ab52587 (1/200 dilution), ab182422 (1/300 dilution), ab92511 (1/200 dilution), ab133616 (1/600 dilution), ab101500 (1/300 dilution), ab9475 (1/100 dilution), ab192847 (1/300 dilution), ab52625 (1/400 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

Image acquisition was performed with TissueFAXS Spectra (TissueGnostics).

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.

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.

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 Alexa Fluor^® 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).

Alexa Fluor^® 488 (ab192643) and Alexa Fluor^® 647 (ab192980) conjugated versions are available for this clone.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab52625 staining Cytokeratin 19in 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). 

Alexa Fluorr^® 488 (ab192643) and Alexa Fluorr^® 647 (ab192980) conjugated versions are available for this clone. 

Unpurified ab52625 showing positive staining in Breast carcinoma tissue.

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

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.

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. 

Unpurified ab52625 showing positive staining in Endometrial carcinoma tissue.

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

Unpurified ab52625 showing negative staining in Glioma tissue.

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

Unpurified ab52625 showing positive staining in Gastric adenocarcinoma tissue.

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