Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).

 

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences®).

 

The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.

 

Sodium citrate antigen retrieval (Leica ER1, pH6.0, 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

 

DAPI (dark blue) was used as a nuclear counter stain.

 

Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.

ab7753 staining human skin sections by IHC-P. The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Blocking of the sample was done with 1% BSA for 10 minutes at 21°C, followed by staining with ab7753 at 1/250 in TBS/BSA/azide for 2h at 21°C. A biotinylated goat anti-rabbit polyclonal antibody at 1/200 was used as the secondary antibody.

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Anti-pan Cytokeratin antibody [C-11] (ab7753) + human A431 cell line on 10% gel.


This is a pan cytokeratin antibody and there are several cytokeratin bands between about 40 and 55 kDa. The identity of the particular cytokeratin that each band corresponds to has not been determined.

Overlay histogram showing HeLa cells stained with ab7753 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab7753, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

ab7753 staining pan Cytokeratin in Mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in a citric acid. Samples were incubated with primary antibody (1/250 in TBS/BSA/azide) for 2 hours at 21°C. A Biotin-conjugated Goat anti-mouse IgG polyclonal (1/250) was used as the secondary antibody.

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ab7753 staining pan Cytokeratin in guinae pig breast carcinoma tissue section by Immunohistochemistry (Formalin/PFA fixed paraffin-embedded sections).