Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human spleen tissue labelling PDI with ab243644 at 1.02 µg/mL (B), PD-L 1 with ab213524 at 1/100 dilution (C) and CD68 with ab213363 at 1/300 dilution (D). Anti-Rabbit and Mouse Polymer HRP was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. Heat mediated antigen retrieval (Leica ER2, PH9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibodies from the previous round, to avoid any cross-reactivity. 

Panel A: merged staining of anti- PD1 (green, Opal™520), anti- PD-L1 (red, Opal™570) and anti- CD68 (yellow, Opal™690).

Panel B: Anti- PD1 stained on antigen-stimulated T cells.

Panel C: anti- PD-L1 stained on cells involved in T cell inhibition

Panel D: anti-CD68 stained on macrophages.

The section was incubated in three rounds of staining: in the order of ab243644, ab213363 and ab213524 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.

All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution

Lane 1 : Wild-type A549 treated with 100 ng/mL IFN gamma (ab259377) for 48 h cell lysate
Lane 2 : CD274 knockout A549 treated with 100 ng/mL IFN gamma (ab259377) for 48 h cell lysate
Lane 3 : U-87 MG cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : Wild-type A549 untreated cell lysate
Lane 6 : CD274 knockout A549 untreated cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab213524 Recombinant Anti-PD-L1 antibody [EPR19759] was shown to specifically react with PD-L1 in wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell line ab267054 (treated and untreated knockout cell lysates ab256831) were used. Wild-type and PD-L1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling PD-L1 with ab213524 at a dilution of 1:250. Heat mediated antigen retrieval was performed using AR9 antigen retrieval solution, and microwave treatment for 15 min at 20% power. Anti-Rabbit/Mouse HRP polymer (PerkinElmer Opal Polymer HRP Ms Plus Rb) was used as secondary antibody. Opal tyramide amplification was performed using Opal 520 fluorophore. Counterstained with DAPI stain. Image scanned with Vectra 3.0 and analyzed via Phenochart software.
This image was courteously provided by Dr. Houssein Abdul Sater, Georgia Cancer Center.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling PD-L1 with ab213524 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Membrane staining on the human tonsil crypt epithelium is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO (Chinese hamster ovary cell line) cells labeling PD-L1 with ab213524 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membrane and cytoplasmic staining on CHO-PDL1 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab213524 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution

Lane 1 : Wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cell lysate
Lane 2 : CD274 knockout A549 treated with 100 ng/mL IFN gamma for 48 h cell lysate
Lane 3 : U-87 MG cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : Wild-type A549 untreated cell lysate
Lane 6 : CD274 knockout A549 untreated cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab213524 Recombinant Anti-PD-L1 antibody [EPR19759] was shown to specifically react with PD-L1 in wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell line ab267055 (treated and untreated knockout cell lysates ab256866) were used. Wild-type and PD-L1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution

Lane 1 : H1975 (Human non-small cell lung cancer epithelial cell) whole cell lysate
Lane 2 : NCI-H1299 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 3 : A549 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 4 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 5 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : SK-BR-3 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 7 : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 8 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate
Lane 9 : PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate
Lane 10 : DU 145 (Human prostate carcinoma epithelial cell) whole cell lysate
Lane 11 : A375 (Human malignant melanoma epithelial cell) whole cell lysate
Lane 12 : MeWo (Human malignant melanoma fibroblast) whole cell lysate
Lane 13 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 14 : Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 15 : HepG2(Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 16 : BXPC-3 (Human pancreas adenocarcinoma epithelial cell) whole cell lysate
Lane 17 : PANC-1 (Human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate
Lane 18 : NIH:OVCAR-3 (Human ovary adenocarcinoma epithelial cell) whole cell lysate
Lane 19 : SK-OV-3 (Human ovarian cancer epithelial cell) whole cell lysate
Lane 20 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 33 kDa
Observed band size: 40-60 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

Expression of PD-L1 varied widely among the tumor cell lines

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 0.5 µg/ml

Lane 1 : Untreated A549 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 2 : A549 (Human lung carcinoma epithelial cell) treated with 100ng/ml Interferon gamma for 48 hours whole cell lysate

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

Predicted band size: 33 kDa


Exposure time: 3 seconds

Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of CHO-PD-L1 (red) and CHO-S (blue) cells labelling PD-L1 with ab213524 at 1/500. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% tween-20-PBS and blocked with 10% goat serum. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCI-H1975 (Human lung non small cell carcinoma cell line) cells labeling PD-L1 with ab213524 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing weakly membrane and cytoplasmic staining on NCI-H1975 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab213524 at 1/100 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling PD-L1 with ab213524 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Membrane staining on the human placenta is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (ab209101)

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

Immunohistochemical analysis of paraffin-embedded human stomach cancer tissue labeling PD-L1 with ab213524 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Membrane staining on the human stomach cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution

Lane 1 : Chinese hamster ovary cell lysate
Lane 2 : Chinese hamster ovary cell lysate overexpressing PD-L1

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 33 kDa
Observed band size: 40-60 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The lower band is predicted to be isoform 2.

Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution + NCI-H1975 (Human non-small cell lung cancer cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa
Observed band size: 40-60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 26546452).

PD-L1 was immunoprecipitated from 0.35 mg of NCI-H1975 (Human non-small cell lung cancer cell line) whole cell lysate with ab213524 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab213524 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1: NCI-H1975 whole cell lysate 10µg (Input).

Lane 2: ab213524 IP in NCI-H1975 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab213524 in NCI-H1975 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.