Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Mouse IgG1 staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279292 was shown to bind specifically to PD-L1. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line ab267054 (knockout cell lysate ab256831). To generate this image, wild-type and Cd274 knockout A549 cell lysates were analysed. 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 2:

Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (ab279292) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

Lane 1:

Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg

Lane 2:

CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 33 kDa

Observed band size: 48 kDa

Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

Lane 1: CHO-S (Chinese hamster ovary epithelial cell) whole cell lysate at 20 µg

Lane 2: CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa

Exposure time: 3s

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237726).

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

(IHC image of ab237726 staining PD-L1 in PD-L1 Dynamic Range Analyte Control formalin fixed paraffin embedded cell lines HistoCyte Laboratories), performed on a Leica BOND RX (standard Protocol F, Polymer Refine kit). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 30 mins at 98°C. The section was then incubated with ab237726, 1μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system for 8 minutes at room temperature. DAB was used as the chromogen for 10 minutes at room temperature. The section was then counterstained with hematoxylin, blued, dehydrated, cleared and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [CAL10] (ab237726)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) and CHO-S (chinese hamster ovary epithelial cell) cells labeling PD-L1 with ab237726 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining in CHO-PD-L1 cells. The nuclear counter stain is DAPI (blue). Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at a 1/200 dilution (red). The negative control is the secondary antibody only.

Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

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 epsilon (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.

Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literature (PMID: 26546452).

All lanes: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

Lane 1: A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2: A549 treated with 100ng/ml IFN gamma for 48h whole cell lysate at 20 µg

Secondary

All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa

Exposure time: 26s

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab237726).