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.

 

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

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 control: PBS instead of the primary antibody.

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 

Representative images of PD-L1 expression in human lung adenocarcinoma and squamous cell carcinoma specimens.

(A) B) 1.0–4.9%, (C) 5.0–9.9%, (D) 10.0–49.9%, and (E) ≥50.0% PD-L1-positive cells.

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

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.

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).

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.

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

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).

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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling PD-L1 with ab205921 on Ventana Ultra. Tumor cells and immune cells show PD-L1 positive 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human non-small cell lung cancer tissue labelling PD-L1 with ab205921. Staining can be seen in tumor associated macrophages and tumor cells.

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).

Immunohistochemical analysis of Human Tonsil tissue with ab205921 at 2 µg/ml.
PD-L1 positive expression of the crypt epithelium (large black arrow) and cells localized within the germinal centers (small black arrow)
Note negative staining of the stroma (red arrow), additionally, stainings of follicles and some interfollicular cells

 

For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

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

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 K.O. cell line.

 

For recommended Flow Cytometry (Flow Cyt) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

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

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 protocol section and/or here (downloadable copy).

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

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 (1,3,4,5,6) 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 protocol section and/or here (downloadable copy).

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

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 protocol section and/or here (downloadable copy).

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

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 (1,3,4,5,6,7) 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 protocol section and/or here (downloadable copy).

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

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human tonsil 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).

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).

Immunohistochemical analysis of formalin-fixed, paraffin-embedded PD-L1 negative Non-small cell lung carcinoma (NSCLC) tissue 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).

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).

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.

Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889) + NCI-H1975 (human non-small cell lung cancer) whole cell lysate at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

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 buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST

All lanes : Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889)

Lane 1 : CHO-S cell lysate
Lane 2 : Human PD-L1 transfected CHO-S cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

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


Exposure time: 1 minute

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST