Anti-PD-L1 抗体 [EPR19759] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

Tissue Microarrays stained for "Anti-PD-L1 antibody [EPR19759]" using "ab213524"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 perform heat mediated antigen retrieval before commencing with IHC staining protocol. The sections were incubated with ab213524 at +4°C overnight. The secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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] - BSA and Azide free (AB221612)

This IHC data was generated using the same anti-PDL1 antibody clone, EPR19759, in a different buffer formulation (cat# ab213524).

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.

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] - BSA and Azide free (AB221612)

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] - BSA and Azide free (AB221612)

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.

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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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 Detection Reagent (HRP) (ab131366), was used for detection 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='/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] - BSA and Azide free (AB221612)

This data was developed using the same antibody clone in a different buffer formulation (ab213524).

Lanes 1 - 6 : Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab213524 Recombinant Anti-PD-L1 antibody [EPR19759] was shown to specifically react with PD-L1 in wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell line ab267054 (treated and untreated knockout cell lysates ab256831) were used. Wild-type and PD-L1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lane 1:

Wild-type A549 treated with 100 ng/ml IFN gamma (<a href='/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 48 h cell lysate at 20 µg

Lanes 2 and 6:

CD274 knockout A549 treated with 100 ng/ml IFN gamma (<a href='/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 48 h cell lysate at 20 µg

Lane 2:

Western blot - Human CD274 (PD-L1) knockout A549 cell line (<a href='/products/cell-lines/human-cd274-pd-l1-knockout-a549-cell-line-ab267054'>ab267054</a>)

Lane 3:

U-87 MG cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Lane 5:

Wild-type A549 untreated cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

This data was developed using the same antibody clone in a different buffer formulation (ab213524).

Lanes 1 - 6 : Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab213524 Recombinant Anti-PD-L1 antibody [EPR19759] was shown to specifically react with PD-L1 in wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell line ab267055 (treated and untreated knockout cell lysates ab256866) were used. Wild-type and PD-L1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (ab221612) at 1/1000 dilution

Lane 1:

Wild-type A549 treated with 100 ng/mL IFN gamma (<a href='/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 48 h cell lysate at 20 µg

Lane 2:

CD274 knockout A549 treated with 100 ng/mL IFN gamma (<a href='/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 48 h cell lysate at 20 µg

Lane 3:

U-87 MG cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Lane 5:

Wild-type A549 untreated cell lysate at 20 µg

Lane 6:

CD274 knockout A549 untreated cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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='/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='/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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

Clone EPR19759 (ab221612) has been successfully conjugated by Abcam. This image was generated using Anti-PD-L1 antibody [EPR19759] (Alexa Fluor® 647). Please refer to ab215251 for protocol details.

ab215251 staining PDL1 in CHO-PDL1 cells. The lower panels demonstrate that ab215251 does not cross react with un-transfected CHO cells.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab215251 at 1/200 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free (AB221612)

Clone EPR19759 (ab221612) has been successfully conjugated by Abcam. This image was generated using Anti-PD-L1 antibody [EPR19759] (Alexa Fluor® 488). Please refer to ab214958 for protocol details.

ab214958 staining PDL1 in CHO-PDL1 cells. The lower panels demonstrate that ab214958 does not cross react with un-transfected CHO cells.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab214958 at 1/200 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).