Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

Immunohistochemical analysis of formalin fixed paraffin embedded Human tonsil labelling PD L1 with ab213524 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab213524 anti PD L1 antibody EPR19759 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

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

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

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

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

• IP

Supplier Data

Immunoprecipitation - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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 secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/10000 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

All lanes:

Immunoprecipitation - Anti-PD-L1 antibody [EPR19759] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-epr19759-ab213524'>ab213524</a>)

Predicted band size: 33 kDa

false

• WB

Lab

Western blot - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

ab228415 works better than ab213524 in western blot testing. We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results. Blocking/Diluting buffer and concentration : 5% NFDM/TBST This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

All lanes:

Western blot - Anti-PD-L1 antibody [EPR19759] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-epr19759-ab213524'>ab213524</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Lane 4:

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PD-L1 antibody [EPR19759] - Low endotoxin, Azide free (AB246695)

Intracellular 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213524).