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    pd-l1-antibody-73-10-ab228415.pdf

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RecombinantRabMAb

Recombinant Anti-PD-L1 antibody [73-10] (ab228415)

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Flow Cytometry (Intracellular) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [73-10] (ab228415)
  • Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Frozen sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
  • Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
  • Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
  • Immunoprecipitation - Anti-PD-L1 antibody [73-10] (ab228415)
  • Anti-PD-L1 antibody [73-10] (ab228415)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [73-10] to PD-L1
  • Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cyt (Intra), IHC-Fr
  • Reacts with: Human, Recombinant fragment

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 594 Alexa Fluor® 647 Carrier Free

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Overview

  • Product name

    Anti-PD-L1 antibody [73-10]
    See all PD-L1 primary antibodies
  • Description

    Rabbit monoclonal [73-10] to PD-L1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cyt (Intra), IHC-Frmore details
  • Species reactivity

    Reacts with: Human, Recombinant fragment
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Chinese hamster ovary cell lysate stably expressing PD-L1; MDA-MB-231, H1975, NCI-H1299, A549, PC3, DU145, A375, U-87 MG, BXPC-3, NIH:OVCAR-3, SK-OV-3, and HeLa whole cell lysates; Human thymus and placenta tissue lysate. IHC-P: Human placenta, lung carcinoma and tonsil tissues. ICC/IF: CHO-PD-L1 cells. Flow Cyt (intra): CHO-PD-L1 cells. IP: NCI-H1975 whole cell lysate. IHC-Fr: Frozen human tonsil tissue sections
  • General notes

    Clone 73-10 is also known as clone MKP1A07310. 

    Clone 73-10 has been tested within Blueprint Phase 2 project. 

    See here for more details.

    Ab228415 (PD-L1 clone 73-10) is a catalogue antibody for Research Use Only. Not for use in diagnostic procedures.

    PBS only (BSA/Azide free) version available - see Ab226766

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    73-10
  • Isotype

    IgG
  • Research areas

    • Immunology
    • Cell Type Markers
    • CD
    • Non-lineage
    • Immunology
    • Immune System Diseases
    • Autoimmune
    • Cancer
    • Tumor immunology
    • Tumor-associated antigens
    • Cancer
    • Tumor biomarkers
    • Tumor antigens

Associated products

  • Alternative Versions

    • Anti-PD-L1 antibody [73-10] - BSA and Azide free (ab226766)
    • Alexa Fluor® 488 Anti-PD-L1 antibody [73-10] (ab237402)
    • Alexa Fluor® 647 Anti-PD-L1 antibody [73-10] (ab237403)
    • Anti-PD-L1 antibody [73-10] - Low endotoxin, Azide free (ab246698)
    • Alexa Fluor® 555 Anti-PD-L1 antibody [73-10] (ab274896)
    • Alexa Fluor® 594 Anti-PD-L1 antibody [73-10] (ab275355)
  • Compatible Secondaries

    • VeriBlot for IP Detection Reagent (HRP) (ab131366)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant human PD-L1 protein (Active) (ab167713)
  • Related Products

    • Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602)
    • Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (ab195889)
    • Universal HIER antigen retrieval reagent (10X) (ab208572)
    • Rabbit specific IHC polymer detection kit HRP/DAB (ab209101)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab228415 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P (1)
1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Recommended incubation time: 10 mins at room temperature

 

WB
1/1000. Detects a band of approximately 40-60 kDa (predicted molecular weight: 33 kDa).
ICC/IF
1/200.
IP
1/30.
Flow Cyt (Intra)
1/100.
IHC-Fr
Use a concentration of 0.05 µg/ml.
Notes
IHC-P
1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Recommended incubation time: 10 mins at room temperature

 

WB
1/1000. Detects a band of approximately 40-60 kDa (predicted molecular weight: 33 kDa).
ICC/IF
1/200.
IP
1/30.
Flow Cyt (Intra)
1/100.
IHC-Fr
Use a concentration of 0.05 µg/ml.

Target

  • Function

    Involved in the costimulatory signal, essential for T-cell proliferation and production of IL10 and IFNG, in an IL2-dependent and a PDCD1-independent manner. Interaction with PDCD1 inhibits T-cell proliferation and cytokine production.
  • Tissue specificity

    Highly expressed in the heart, skeletal muscle, placenta and lung. Weakly expressed in the thymus, spleen, kidney and liver. Expressed on activated T- and B-cells, dendritic cells, keratinocytes and monocytes.
  • Sequence similarities

    Belongs to the immunoglobulin superfamily. BTN/MOG family.
    Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.
  • Cellular localization

    Cell membrane and Endomembrane system.
  • Target information above from: UniProt accession Q9NZQ7 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 29126 Human
    • Omim: 605402 Human
    • SwissProt: Q9NZQ7 Human
    • Unigene: 521989 Human
    • Alternative names

      • B7 H antibody
      • B7 H1 antibody
      • B7 homolog 1 antibody
      • B7-H1 antibody
      • B7H antibody
      • B7H1 antibody
      • CD 274 antibody
      • CD-274 antibody
      • CD274 antibody
      • CD274 antigen antibody
      • CD274 molecule antibody
      • MGC142294 antibody
      • MGC142296 antibody
      • OTTHUMP00000021029 antibody
      • PD L1 antibody
      • PD-L1 antibody
      • PD1L1_HUMAN antibody
      • PDCD1 ligand 1 antibody
      • PDCD1L1 antibody
      • PDCD1LG1 antibody
      • PDL 1 antibody
      • PDL1 antibody
      • Programmed cell death 1 ligand 1 antibody
      • Programmed death ligand 1 antibody
      • RGD1566211 antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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 Cytometry (Intracellular) - Anti-PD-L1 antibody [73-10] (ab228415)
      Flow Cytometry (Intracellular) - Anti-PD-L1 antibody [73-10] (ab228415)

      Intracellular 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.

    • Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [73-10] (ab228415)

      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.

    • Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
      Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
      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.

    • Immunohistochemistry (Frozen sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Frozen sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      IHC image of PD-L1 staining in a section of frozen normal human tonsil* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab228415, 0.05ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.


      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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)Image from Silva MA et al., PLoS One. 2018;13(6):e0196464. Fig 3(B).; 10.1371/journal.pone.0196464.

      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) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)Image from Tsao MS et al., J Thorac Oncol. 2018;13(9):1302-1311. Fig 3.; 10.1016/j.jtho.2018.05.013 with permission from Elsevier.

      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) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)Image from Silva MA et al., PLoS One. 2018;13(6):e0196464. Fig 4.; 10.1371/journal.pone.0196464. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

      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.

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [73-10] (ab228415)

      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).

    • Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
      Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
      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.

    • Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
      Western blot - Anti-PD-L1 antibody [73-10] (ab228415)
      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).

    • Immunoprecipitation - Anti-PD-L1 antibody [73-10] (ab228415)
      Immunoprecipitation - Anti-PD-L1 antibody [73-10] (ab228415)

      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.

    • Anti-PD-L1 antibody [73-10] (ab228415)
      Anti-PD-L1 antibody [73-10] (ab228415)

    Protocols

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    References (22)

    Publishing research using ab228415? Please let us know so that we can cite the reference in this datasheet.

    ab228415 has been referenced in 22 publications.

    • Yoshikawa K  et al. Prognostic significance of PD-L1-positive cancer-associated fibroblasts in patients with triple-negative breast cancer. BMC Cancer 21:239 (2021). PubMed: 33676425
    • Rösner E  et al. Prognostic value of PD-L1 expression in bronchopulmonary neuroendocrine tumours. Endocr Connect 10:180-190 (2021). PubMed: 33475525
    • Li F  et al. POU2F1 induces the immune escape in lung cancer by up-regulating PD-L1. Am J Transl Res 13:672-683 (2021). PubMed: 33594317
    • Mei J  et al. A comparability study of natural and deglycosylated PD-L1 levels in lung cancer: evidence from immunohistochemical analysis. Mol Cancer 20:11 (2021). IHC-P ; Human . PubMed: 33413365
    • Fu R  et al. Prognostic Significance of Serum PD-L1 Level in Patients with Locally Advanced or Metastatic Esophageal Squamous Cell Carcinoma Treated with Combination Cytotoxic Chemotherapy. Cancer Manag Res 13:4935-4946 (2021). PubMed: 34188546
    View all Publications for this product

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    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-PD-L1 antibody [73-10]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (tonsil)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9.0
    Permeabilization
    No
    Specification
    tonsil
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: RT°C
    Fixative
    10% NBF
    Read More

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    Verified customer

    Submitted Aug 12 2019

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