Anti-PD1 antibody [CAL20]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human tonsil tissue staining PD-L1 with ab205921 at a 1 : 500 (2.19 ug/ml) dilution; PD1 with ab237728 at 1 : 2000 (0.525 ug/ml) dilution and CD68 with ab213363 at 1 : 500 (1.26 ug/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.

Panel A : merged staining of anti-CD68 (magenta; Opal^™690), anti-PD-L1 (green; Opal^™520) and anti-PD1 (red; Opal^™570) on human tonsil.
Panel B : anti-PD-L1 stained on cells involved in T cell inhibition.
Panel C : anti-PD1 stained on antigen-stimulated T cells.
Panel D : anti-CD68 stained on macrophages.

The section was incubated in three rounds of staining : in the order of ab213363 and ab205921 for 30 mins, then ab237728 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (AB237728)

Formalin-fixed, paraffin-embedded human lung carcinoma tissue stained for PD1 using ab237728 at 0.125 μg/ml dilution in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (AB237728)

Formalin-fixed, paraffin-embedded human lymph node tissue stained for PD1 using ab237728 at 0.125 μg/ml dilution in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (AB237728)

Immunohistochemical analysis of paraffin-embeded human tonsil tissue labeling PD1 with ab237728 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the human tonsil.is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins.

The section was incubated with ab237728 for 15 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-PD1 (ab237728; orange; Opal™520) anti-PDL1 (ab237726; green; Opal™540) anti-CD68 (ab192847; yellow; Opal™570) anti-CD3 epsilon (ab16669; red; Opal™620) anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT Akoya Biosciences^®).

The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (Leica ER1 pH6.0 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round to avoid any cross-reactivity.

DAPI (dark blue) was used as a nuclear counter stain.

Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD1 antibody [CAL20] (AB237728)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MOLT-4 (human lymphoblastic leukemia cell line) cells labeling PD1 with ab237728 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in MOLT-4 cells treated with PMA (10ng/ml 24h) and Ionomycin(500ng/ml 24h). The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Cells were either untreated, ot treated with treated with PMA (10ng/ml 24h) and Ionomycin (500ng/ml 24h).

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (AB237728)

The BOND™ Polymer Refine Detection System on the BOND RX Automated Stainer (DS9800, Leica Biosystems, Wetzlar, Germany) was used for immunohistochemical labelling of formalin-fixed, paraffin-embedded (FFPE) tissues. The protocol includes a peroxide block, post-primary reagent, polymer detection, DAB chromogen, and haematoxylin counterstain, all automated to minimize variability. Antigen retrieval was performed by heat-induced epitope retrieval (HIER) for 20 minutes in Tris-EDTA buffer (BOND Epitope Retrieval Solution 2, AR9640, Leica Biosystems, Wetzlar, Germany). For mouse and rabbit antibodies, the sequence comprised peroxide block (10 min), primary antibody incubation (30 min), post-primary (10 min), polymer (10 min), DAB (10 min), and haematoxylin (8 min). For rat antibodies, an anti-rat step (1 : 300 dilution, Vector Laboratories, California, USA; 20 min) was included prior to polymer application (15 min).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (AB237728)

Tissue Microarrays stained for "Anti-PD1 antibody [CAL20]" using "ab237728"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 Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 30 minutes. The sections were incubated with ab237728 for 15 mins at room temperature followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (AB237728)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• WB

Lab

Western blot - Anti-PD1 antibody [CAL20] (AB237728)

False colour image of Western blot : Anti-PD1 antibody [CAL20] staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab237728 was shown to bind specifically to PD1. A band was observed at 48 kDa in wild-type HeLa cell lysates with no signal observed at this size in PDCD1 CRISPR-Cas9 edited cell line ab255417 (CRISPR-Cas9 edited cell lysate ab263794). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of PD1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and PDCD1 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PD1 antibody [CAL20] (ab237728) at 1/500 dilution

Lane 1:

Wild-type HeLa Vehicle Control lonomycin (0 ng/mL, 24h) + PMA (10 ng/mL, 24 h) cell lysate at 20 µg

Lane 2:

Wild-type HeLa Treated lonomycin (500 ng/mL, 24h) + PMA (10 ng/mL, 24 h) cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human PDCD1 (PD1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-pdcd1-pd1-knockout-hela-cell-line-ab255417'>ab255417</a>)

Lane 3:

PDCD1 knockout HeLa vehicle Control lonomycin (0ng/mL 24h) + PMA (10ng/mL 24h) cell lysate at 20 µg

Lane 4:

PDCD1 knockout HeLa lonomycin (500ng/mL 24h) + PMA (10ng/mL 24h) cell lysate at 20 µg

false

• WB

Supplier Data

Western blot - Anti-PD1 antibody [CAL20] (AB237728)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PD1 antibody [CAL20] (ab237728) at 1/1000 dilution

Lane 1:

Untreated MOLT-4 (human lymphoblastic leukemia cell line), whole cell lysate at 20 µg

Lane 2:

MOLT-4 (treated with 10ng/ml PMA and 500ng/ml Ionomycin for 24h), 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 1/100000 dilution

Predicted band size: 32 kDa

Observed band size: 50-55 kDa

false

Exposure time: 3min

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (AB237728)

Immunohistochemical analysis of paraffin-embeded Rhesus monkey tonsil tissue labeling PD1 with ab251613 followed by Polink 1 Polymer HRP anti-Rabbit IgG.

Heat mediated antigen retrieval-Buffer/Enzyme Used : Dako pH9.

This data was developed using the same antibody clone in a different buffer formulation (ab251613).

This image is courtesy of Dr. Chi Ngai Chan