Name: Anti-PD1 antibody [CAL20]
SKU: ab237728
Rating: 5 (1 reviews)

Anti-PD1 antibody [CAL20] (ab237728) is a rabbit monoclonal antibody that is used to detect PD1 in Western Blot, IHC-P, ICC/IF, mIHC. Suitable for Human, Rhesus monkey samples.

- Validated for multiplex IHC on the Leica BOND® MAX using Opal reagents

-Using biophysical QC, antibody identity is confirmed at a molecular level for unrivalled batch-batch consistency

Images

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

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.05% Sodium azide
Constituents: PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

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Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	Flow Cyt	WB	mIHC	ICC/IF	IHC-P
Human	Not recommended	Not recommended	Tested	Tested	Tested	Tested
Rhesus monkey	Not recommended	Not recommended	Expected	Expected	Expected	Tested

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rhesus monkey	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human, Rhesus monkey	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rhesus monkey	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rhesus monkey	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/50	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rhesus monkey	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rhesus monkey	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Associated Products

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Target data

See full target information PDCD1

Function

The protein expressed by the gene PDCD1 functions as an inhibitory receptor on antigen-activated T-cells, playing a critical role in the induction and maintenance of immune tolerance to self. It delivers inhibitory signals by binding to the ligands CD274/PDCD1L1 and CD273/PDCD1LG2. Tumors exploit the PDCD1-mediated inhibitory pathway to attenuate anti-tumor immunity, enabling tumor survival by evading immune system destruction. The interaction with CD274/PDCD1L1 inhibits the effector function of cytotoxic T lymphocytes (CTLs). Blocking the PDCD1-mediated pathway can reverse the exhausted T-cell phenotype and normalize the anti-tumor response, providing a rationale for cancer immunotherapy. This supplementary information is collated from multiple sources and compiled automatically.

Alternative names

CD279, PD1, PDCD1, Programmed cell death protein 1, Protein PD-1, hPD-1

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.05% Sodium azide
Constituents: PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: CAL20
Purification technique: Affinity purification Protein A
Concentration
Purification notes: Purity >99%

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

What is this antibody validated in?

Anti-PD1 antibody [CAL20] (ab237728) is a rabbit recombinant monoclonal antibody and is validated for use in Western Blot (WB), Immunohistochemistry (IHC-P), Immunocytochemistry/immunofluorescence (ICC/IF), Multiplex IHC (mIHC) in Human, Rhesus monkey samples.

What is the molecular weight of PD1?

Anti-PD1 [CAL20] (ab237728) specifically detects a band for PD1 (UniProt: Q15116) at a molecular weight of 32kDa.

Trusted by the scientific community

Anti-PD1 [CAL20] (ab237728) was first used in a scientific publication in 2018 and has been cited over 10 times in peer-reviewed journals.

Trial sizes available!

Test your antibody or perform pre-screening before committing to a larger quantity. Sold in 10µl. Discover our selection of trial-size antibodies.

Other related products

We have a range of other formats of antibody clone [CAL20] also available for your convenience:
ab237728, Carrier free - ab251613, ab300009

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

PD1 also known as Programmed Cell Death Protein 1 or PDCD1 is a transmembrane protein that plays a critical role in regulating immune responses. It has a mass of approximately 55 kDa. PD1 is expressed on the surface of T cells B cells and some myeloid cells. PD1’s expression increases upon activation of these immune cells assisting in maintaining peripheral tolerance. Researchers often use PD1 mouse models and chimeric antibodies to explore the function of PD1 for experimental purposes. Antibodies such as anti-PD1 such as EH12.2H7 help in blocking PD1 interaction to study its role further.

Biological function summary

PD1 serves as an inhibitory receptor acting as a checkpoint in the immune system. It becomes part of an immune-suppressive complex when it binds with its ligands PD-L1 or PD-L2 which are expressed on various cell types including some tumor cells. This interaction suppresses the proliferation of T cells and cytokine production contributing to immune homeostasis. By controlling T cell activity PD1 limits autoimmunity but can also reduce the immune system's capability to attack cancer cells.

Pathways

PD1 functions in the immune checkpoint pathway a critical regulatory circuit in immune regulation. The engagement of PD1 with its ligands initiates a cascade that inhibits the function and proliferation of T cells through downstream SHP-2 phosphatase activity. This pathway frequently involves other regulatory proteins like CTLA-4 and is an important mechanism by which the body modulates immune responses. Related pathways often intersect with those involving T cell receptor signaling and contribute to the overall modulation of immune activity.

Associated diseases and disorders

PD1 has a significant role in cancer and autoimmune disorders. PD1 expression can allow tumors to evade immune surveillance making PD1 a target for cancer therapies such as anti-PD1 antibodies which aim to block PD1 and restore T cell activity. The interaction of PD1 with cancer-related proteins like PD-L1 facilitates tumor immune evasion. In autoimmune disorders PD1’s regulation of immune balance can become dysregulated leading to persistent immune activation and tissue damage. Understanding PD1 and its interaction with proteins such as PD-L1 helps in developing therapeutic strategies for both cancer and autoimmune conditions.

Product promise

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In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

15 product images

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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 32 kDa
Observed band size: 50-55 kDa
Exposure time: 3min
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.
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) (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) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (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).
Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (ab237728)
PD1 Multiplex immunohistochemistry staining of human tonsil tissue using rabbit Anti-PD1 antibody
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 (Anti-PD-L1 antibody [CAL10] ab237726; green; Opal™540) anti-CD68 (Anti-CD68 antibody [SP251] ab192847; yellow; Opal™570) anti-CD3 epsilon (Anti-CD3 epsilon antibody [SP7] ab16669; red; Opal™620) anti-Ki67 (Anti-Ki67 antibody [SP6] ab16667; light blue; Opal™650) and anti-PanCK (Anti-pan Cytokeratin antibody [C-11] 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), Anti-PD-L1 antibody [CAL10] ab237726 (1/500 dilution), Anti-CD68 antibody [SP251] ab192847 (1/300 dilution), Anti-CD3 epsilon antibody [SP7] ab16669 (1/300 dilution), Anti-Ki67 antibody [SP6] ab16667 (1/200 dilution) and Anti-pan Cytokeratin antibody [C-11] 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.
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.
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.
This image is courtesy of Dr. Chi Ngai Chan
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [CAL20] (ab237728)
Immunohistochemical analysis of paraffin-embeded Rhesus monkey tonsil tissue labeling PD1 with Anti-PD1 antibody [CAL20] - BSA and Azide free 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 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613).
This image is courtesy of ImmunoAtlas.
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 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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 Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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.
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 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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 Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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.
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.
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] (Anti-GAPDH antibody [6C5] - Loading Control 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 Human PDCD1 (PD1) knockout HeLa cell line ab255417 (CRISPR-Cas9 edited cell lysate Human PDCD1 (PD1) knockout HeLa 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 (Human PDCD1 (PD1) knockout HeLa cell line ab255417)
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
Performed under reducing conditions.
Multiplex immunohistochemistry - Anti-PD1 antibody [CAL20] (ab237728)
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human tonsil tissue staining PD-L1 with Anti-PD-L1 antibody [28-8] ab205921 at a 1:500 (2.19 ug/ml) dilution; PD1 with ab237728 at 1:2000 (0.525 ug/ml) dilution and CD68 with Anti-CD68 antibody [EPR20545] 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 Anti-CD68 antibody [EPR20545] ab213363 and Anti-PD-L1 antibody [28-8] 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
This image is courtesy of ImmunoAtlas.
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 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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 Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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.
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 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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 Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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.
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 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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 Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free 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.