Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Tissue Microarrays stained for Anti-PD-L1 antibody [28-8] using ab205921 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent). The sections were incubated with ab205921 at +4°C overnight. For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

• ICC/IF

AbReview61595****

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Paraformaldehyde-fixed Triton X-100 permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell line) cells stained for PD-L1 (red) using ab205921 at 1/200 dilution in ICC/IF followed by CF568 Donkey anti-rabbit IgG(H+L) secondary antibody at 1/500 dilution.

Alexa Fluor^® 488 (ab209959) and Alexa Fluor^® 647 (ab209960) conjugated versions are available for this clone.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

This image is courtesy of an Abreview submitted by Dr. Dimitra Kalamida.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human non-small cell lung cancer tissue labelling PD-L1 with ab205921. Tumor cells and immuno cells localized within the stroma show PD-LA plasma membrane staining.

For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• Flow Cyt

Collaborator

Flow Cytometry - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

ab205921 specificity testing by Flow Cytometry (KO testing) : Loss of detection on KO cells.Strong detection with anti-PD-L1 (ab205921 clone 28-8) TALEN constructs targeting exon4 of human PD-L1 transcript variant 1 (NM_014143.3) and complete knock out (K.O) confirmed by deep sequencing in clone L2-14. Cell surface staining is almost completely eliminated in the L2987 L2-14 KO cell line. For recommended Flow Cytometry (Flow Cyt) protocol please refer to the protocol book in the protocol section. Alexa Fluor^® 488 (ab209959) and Alexa Fluor^® 647 (ab209960) conjugated versions are available for this clone. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling PD-L1 with ab205921. Tumor cells show weak and partial postive PD-L1 expresseion in the plasma membrane. PD-L1 positive tumor associated immunoe cells are also stained.

For antigen retrival buffer Universal HIER antigen retrieval reagent (ab208572) was used.
For IHC detection kit Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Paraformaldehyde-fixed paraffin-embedded human placenta tissue stained for PD-L1 using ab205921 at 1/100 dilution in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

This data was developed using ab205921, the same antibody clone in a different buffer formulation
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 ab205921 1ugml 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

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of Human Lung NSCLC with ab205921 at 2 μg/ml.
High power view
A) Rabbit IgG 5 μg/mL. No staining
B) Anti PD-L1 2 μg/mL (ab205921 batches 1)
C) Anti PD-L1 2 μg/mL (ab205921 batches 3)
D) Anti PD-L1 2 μg/mL (ab205921 batches 4)
E) Anti PD-L1 2 μg/mL (ab205921 batches 5)
F) Anti PD-L1 2 μg/mL (ab205921 batches 6)

All batches/lots (13456) showed consistent results.

Note linear and complete or partial (arrows) PD-L1 staining of tumor cells. Tumor associated immune cells localized over the tumor margin exhibit positive plasma membrane staining (small arrows).

For recommended Immunohistochemistry (IHC) protocol please refer to the protocol book in the Product Protocol section of ab205921.

This data was developed using the same antibody clone in a different buffer formulation containing PBS BSA glycerol and sodium azide (ab205921).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Anti-PD-L1 antibody [28-8] (ab205921)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling PD-L1 with ab205921 at a dilution of 1 : 400. 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

IHC image of ab205921 staining PD-L1 in human tonsil formalin fixed paraffin embedded tissue sections* performed on a Leica BOND RX (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 ab205921 5μg/ml working concentration for 60 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

This image was generated using ab205921, the same antibody but with BSA and Azide

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of formalin-fixed paraffin-embedded L2987 (Human lung adenocarcinoma cell line with endogenous PD-L1 expression) cells labeling PD-L1 with ab205921 at 2 μg/ml. Counterstained with Hematoxylin.

For antigen retrival buffer Universal HIER antigen retrieval reagent (ab208572) was used.
For IHC detection kit Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of formalin-fixed paraffin-embedded human placenta labelling PD-L1 with ab205921 at a dilution of 2µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab205921 anti PD-L1 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

This data was developed using ab205921, the same antibody clone in a different buffer formulation.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human head and neck squamous cell carcinoma tissue labeling PD-L1 with ab205921 at 2 µg/ml. Counterstained with Hematoxylin.

For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of formalin-fixed paraffin-embedded human tonsil labelling PD-L1 with ab205921 at a dilution of 2µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab205921 anti PD-L1 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

This data was developed using ab205921, the same antibody clone in a different buffer formulation.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

ab205921 specificity testing by Immunohistochemistry (KO testing) : Loss of detection on KO Cells.

Strong IHC detection with anti-PD-L1 (ab205921, clone 28-8) is seen in human lung adenocarcinoma tumor cell line L2987. PDL1 gene was edited in L2987 cells using TALEN constructs targeting exon4 of human PD-L1, transcript variant 1 (NM_014143.3) and complete knock out (K.O) confirmed by deep sequencing in clone L2-14. IHC detection is completely eliminated in the L2987 L2-14 K.O. cell line.

For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the Product Protocol section of ab205921.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• WB

Lab

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

ab228415 works better than ab205921 in western blot testing. Blocking/Diluting buffer and concentration : 5% NFDM/TBST This data was developed using ab205921, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-PD-L1 antibody [28-8] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-28-8-ab205921'>ab205921</a>) at 1/1000 dilution

Lane 1:

MDA-MB-231(human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

U-87 MG (human glioblastoma astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 40-60 kDa

false

Exposure time: 100s

• WB

Unknown

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889)

All lanes:

NCI-H1975 (human non-small cell lung cancer) whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>)

Predicted band size: 33 kDa

Observed band size: 40-60 kDa

false

Exposure time: 3min

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

This data was developed using the same antibody clone in a different buffer formulation (ab205921).
Immunocytochemistry analysis of CHO-PDL1 (PD-L1 stably expessed Chinese hamster ovary epithelial cell) labeling PD-L1 with purified ab205921 at 1/400 dilution. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor^® 488 ab150077) at 1/1000 (2 μg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.32 μg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative controls : Cells not transfected with PD-L1 and both the transfected and mock tranfected cells without the primary antibody.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of CHO Parental cells with ab205921 at 2 μg/ml.
High power view
A) Rabbit IgG 5 μg/mL. No staining
B) Anti PD-L1 2 μg/mL (ab205921 batches 1)
C) Anti PD-L1 2 μg/mL (ab205921 batches 3)
D) Anti PD-L1 2 μg/mL (ab205921 batches 4)
E) Anti PD-L1 2 μg/mL (ab205921 batches 5)
F) Anti PD-L1 2 μg/mL (ab205921 batches 6)
G) Anti PD-L1 2 μg/mL (ab205921 batches 7)

All batches/lots (134567) showed consistent results.

Note absence of PD-L1 expression in CHO parental cells.

For recommended Immunohistochemistry (IHC) protocol please refer to the protocol book in the Product Protocol section.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Immunohistochemical analysis of CHO PD-L1 cells with ab205921 at 2 µg/ml.
High power view
A) Rabbit IgG, 5 µg/mL. No staining
B) Anti PD-L1, 2 µg/mL (ab205921 batches 1)
C) Anti PD-L1, 2 µg/mL (ab205921 batches 3)
D) Anti PD-L1, 2 µg/mL (ab205921 batches 4)
E) Anti PD-L1, 2 µg/mL (ab205921 batches 5)
F) Anti PD-L1, 2 µg/mL (ab205921 batches 6)
G) Anti PD-L1, 2 µg/mL (ab205921 batches 7)

All batches/lots (1,3,4,5,6,7) showed consistent results.

Note strong, moderate, and weak (red, yellow, and white arrows respectively) plasma membrane staining of CHO PD-L1 transfected cells

For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the Product Protocol section of ab205921.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

• IHC-P

AbReview54457****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

This IHC data was generated using the same anti-PDL1 antibody clone, 28-8, in a different buffer formulation (cat# ab205921).

Immunohistochemical staining of PD-L1 in formalin fixed, paraffin embedded human non-squamous non-small cell lung cancer (NSQ-NSCLC) using ab205921 at a dilution of 1/400, incubated for an hour at room temperature. Heat mediated antigen retrieval was carried out in low pH buffer and the sample was blocked with peroxidase blocking buffer for 3 minutes.

This image was courteously provided by Dr. Kai Schmitt from the Institute of Pathology, Saarbrücken-Rastpfuhl.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

IHC image of ab205921 staining PD-L1 in PD-L1 Dynamic Range Analyte Control formalin fixed paraffin embedded cell lines (HistoCyte Laboratories), performed on a Leica BOND RX (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 ab205921, 5μg/ml working concentration, for 60 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.

This image was generated using ab205921, the same antibody but with BSA and Azide

• WB

Lab

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889)

Lane 1:

CHO-S cell lysate at 15 µg

Lane 2:

Human PD-L1 transfected CHO-S cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>)

Predicted band size: 33 kDa

Observed band size: 40-60 kDa

false

Exposure time: 1min

• WB

Collaborator

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (AB209889)

This data was developed using ab205921, the same antibody clone in a different buffer formulation :

(A and B) Western blots of recombinant PD-L1 protein (Lane 1), cell lysates of CHO-PD-L1 (Lane 3), CHO (Lane 4), ES-2 (Lane 5) and Colo205 (Lane 6) cell lines. In B, anti-PD-L1 (ab205921, clone 28-8) was pre-incubated with purified recombinant PDL1 protein overnight at 4°C.

Blank/no sample (Lane2). Lane 2 is blank on purpose.

For recommended Western Blot (WB) protocol, please refer to the protocol book in the Product Protocols section of ab205921

All lanes:

Western blot - Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889)

Predicted band size: 33 kDa

false