IHC image of ab228415 staining PD-L1 in human tonsil formalin fixed paraffin embedded tissue sections*, performed on a Leica BOND RX (standard Protocol F, Polymer Refine kit). The section was pre-treated using heat mediated antigen retrieval with EDTA  buffer (pH9, epitope retrieval solution 2) for 30 mins at 98°C. The section was then incubated with ab228415, 0.06μg/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.

 

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell, Red) / CHO-S (Chinese hamster ovary epithelial cell, Blue) cell lines labeling PD-L1 with ab228415 at 1/100 dilution (red). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) cells labeling PD-L1 with ab228415 at 1/200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution (green). Confocal image showing membranous staining on CHO-PD-L1 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution.

All lanes : Anti-PD-L1 antibody [73-10] (ab228415) at 1/1000 dilution

Lane 1 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : SK-BR-3 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : H1975 (Human non-small cell lung cancer epithelial cell) whole cell lysate
Lane 5 : NCI-H1299 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 6 : A549 (Human lung carcinoma 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 (HRP) (ab97051) at 1/20000 dilution

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


Exposure time: 120 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST. Expression of PD-L1 varied widely among the tumor cell lines.

IHC image of ab228415 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 EDTA buffer (pH9, epitope retrieval solution 2) for 30 mins at 98°C. The section was then incubated with ab228415, 0.06μg/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.

Anti-PD-L1 antibody [73-10 ] (ab228415)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling PD-L1 with ab228415 at a dilution of 1:2500. 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 (Vector Labs) 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 software.
This image was courteously provided by Dr. Houssein Abdul Sater, Georgia Cancer Center.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) of human tonsil staining PD-L1 with ab228415 at 1/500 dilution. Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 10 mins. The section was incubated with ab228415 for 10 mins at room temperature. The secondary antibody used was ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counter stained Hematoxylin. Performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) of human placenta staining PD-L1 with ab228415 at 1/500 dilution. Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 10 mins. The section was incubated with ab228415 for 10 mins at room temperature. The secondary antibody used was ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counter stained Hematoxylin. Performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) of human lung carcinoma staining PD-L1 with ab228415 at 1/500 dilution. Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 10 mins. The section was incubated with ab228415 for 10 mins at room temperature. The secondary antibody used was ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counter stained Hematoxylin. Performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling PD-L1 with ab228415 at 1/5000 dilution. The tissue was incubated with ab228415 at 4℃ overnight. followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Membranous and cytoplasmic staining in human placenta (PMID: 12538684) 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).

Antigen retrieval: Universal HIER antigen retrieval reagent (10X) (ab208572).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) Staining PD-L1 in human non-small cell lung cancer tissue with >50% PD-L1-positive tumor cells were compared with tissue with lower PD-L1 expression using 73-10 at 0.25μg/ml incubated for 30 minutes at room temperature. Antigen Retrieval was done with Target Retrieval Solution, high pH. Detection was done with EnVision FLEX/HRP. Hematoxylin EnVision FLEX was used as a counter stain. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) of lung cancer tissue samples. Comparing the staining PD-L1 with different monoclonal antibodies. 73-10 showed higher sensitivity to PD-L1 compared to the other clones. For further details on this image please see PubMed ID: 29800747.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) Staining PD-L1 in human non-small cell lung cancer tissue using 73-10 at 0.25μg/ml incubated for 30 minutes at room temperature. Antigen Retrieval was done with Target Retrieval Solution, high pH. Detection was done with EnVision FLEX/HRP. Hematoxylin EnVision FLEX was used as a counter stain. 

A: Diffuse expression of PD-L1 (IHC) on tumor cell membranes of a squamous cell carcinoma, including central regions of trabeculae. Prominent labeling of cells in the TME compartment at the tumor-nest-TME interface suggesting presence of an immunological synapse (inset arrow).

B: Patchy expression of PD-L1 in a squamous cell carcinoma at the tumor-nest-TME interface (inset arrow). Minimal to no PD-L1 expression in the trabeculae (asterisk) if compared with (A)

C: No to minimal PD-L1 expression in both tumor and TME compartments in an adenocarcinoma.

D: Diffuse expression of PD-L1 by tumor-nests in an adenocarcinoma with minimal TME staining.

F: TME expression only. No to minimal PD-L1 expression in tumor cells of a squamous cell carcinoma, with widespread staining in the TME compartment.

 

Immunohistochemical staining of PD-L1 in formalin-fixed, paraffin embedded reference standard with negative (-), low positive (+), intermediate positive (++) and strong positive (+++) controlled protein expressing cell lines (‚CD274 (PD-L1) Expression IHC Reference Standard, catalog ID HD787, horizon) using clone 73-10 [ab228415] at a dilution of 2μg/ml. Incubate for 30 minutes at room temperature. Heat mediated antigen retrieval in high pH buffer (Tris/EDTA buffer, pH 9, during 20 min at 95°C). Block sample with peroxidase blocking buffer (EnVision Flex Peroxidase-Blocking Reagent) for 5 minutes. Signal detection with Autostainer Link from Dako and EnVision Flex Kit, High pH (Code K8000).

Immunohistochemical staining of PD-L1 in formalin-fixed, paraffin embedded Formalin-fixed, paraffin-embedded reference standard with negative (-), low positive (+), intermediate positive (++) and strong positive (+++) controlled protein expressing cell lines (‚CD274 (PD-L1) Expression IHC Reference Standard‘, catalog ID HD787, horizon) using clone 73-10 [ab228415] at a dilution of 10μg/ml. Incubate for 30 minutes at 37°C. Heat mediated antigen retrieval in sCC1 (Tris/EDTA buffer, pH 8). Signal detection with BenchMark XT from Roche/Ventana and ultraView Universal DAB Detection Kit (Code 760-500).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling PD-L1 with ab228415 at 1/5000 dilution. The tissue was incubated with ab228415 at 4℃ overnight, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Cytoplasmic and membranous staining in human tonsil 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).

Antigen retrieval: Universal HIER antigen retrieval reagent (10X) (ab208572).

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling PD-L1 with ab228415 at 1/5000 dilution. The tissue was incubated with ab228415 at 4℃ overnight, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Membranous and weakly cytoplasmic staining in human lung carcinoma (PMID: 23460533) 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).

Antigen retrieval: Universal HIER antigen retrieval reagent (10X) (ab208572).

All lanes : Anti-PD-L1 antibody [73-10] (ab228415) at 1/1000 dilution

Lane 1 : CHO-S (Chinese hamster ovary epithelial cell)
Lane 2 : CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell)

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: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PD-L1 antibody [73-10] (ab228415) at 1/1000 dilution

Lane 1 : NCI-H1975 (human non-small cell lung cancer cell line), whole cell lysate
Lane 2 : Human placenta
Lane 3 : Human thymus

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 33 kDa


Exposure time: 70 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular mass 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 ab228415 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab228415 at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution.

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

Lane 2: ab228415 IP in NCI-H1975 whole cell lysate (+). 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab228415 in NCI-H1975 whole cell lysate (-).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 30 seconds.