Name: Anti-CD8 alpha antibody [CAL66]
SKU: ab237709
Rating: 5 (2 reviews)

Anti-CD8 alpha antibody [CAL66] is a rabbit recombinant monoclonal antibody that is used to detect CD8 alpha in Flow cytometry, ICC/IF, IHC-P, IP, mIHC. Suitable for Human, Rat samples.

- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone CAL66 is cited in >70 publications

Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [CAL66] (AB237709), 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	ICC/IF	IHC-P	mIHC
Human	Tested	Tested	Not recommended	Tested	Tested	Tested
Rat	Expected	Expected	Not recommended	Expected	Tested	Expected

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Rat	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.

Expected
Expected

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Rat, Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes -

Expected
Expected

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

Tested
Tested

Species	Dilution info	Notes
Species Rat	Dilution info 1 µg/mL	Notes Use at 1 μg/ml for rat Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 0.25 µg/mL	Notes Use at 1 μg/ml for rat Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

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

Associated Products

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Target data

See full target information CD8A

Function

The protein expressed by the gene CD8A is an integral membrane glycoprotein crucial for immune responses, operating mainly in T-cells as a coreceptor for MHC class I molecule:peptide complexes. These peptides originate from cytosolic proteins, unlike class II peptides, which are from extracellular proteins. CD8A interacts with the T-cell receptor (TCR) and MHC class I proteins on antigen-presenting cells, recruiting the Src kinase LCK to the TCR-CD3 complex. LCK phosphorylates substrates, triggering signaling pathways that lead to the production of lymphokines, enhanced motility, adhesion, and activation of cytotoxic T-lymphocytes (CTLs), thereby aiding in the recognition and elimination of infected or tumor cells. Additionally, in natural killer (NK) cells, CD8A homodimers on the cell surface provide a survival mechanism for conjugating with and lysing multiple target cells. CD8A homodimers also facilitate the survival and differentiation of activated lymphocytes into memory CD8 T-cells. This supplementary information is collated from multiple sources and compiled automatically.

Alternative names

CD8a, MAL, CD8A, T-cell surface glycoprotein CD8 alpha chain, T-lymphocyte differentiation antigen T8/Leu-2

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: CAL66
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

Product Specifications

Anti-CD8 alpha antibody [CAL66] (ab237709) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt, ICC/IF, IHC-P, IP, mIHC in human, rat samples.
Anti-CD8 alpha antibody [CAL66] (ab237709) specifically detects CD8 alpha (UniProt ID: P01732; Molecular weight: 24kDa) and is sold in a convenient trial size to enable initial testing (20 µL) and larger sizes for subsequent scaling up experiments (100 µL and 1 mL).

Quality and Validation

Abcam's high quality manufacturing and validation processes ensure Anti-CD8 alpha antibody [CAL66] (ab237709) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.
Anti-CD8 alpha antibody [CAL66] (ab237709) has been cited over 43 times in peer reviewed journals and is trusted by the scientific community.

Related Products
Conjugation-ready, carrier free format available for antibody clone CAL66 - ab251596.
Antibody clone CAL66 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 647, Alexa Fluor® 488, Alexa Fluor® 555, Alexa Fluor® 594 (ab305048, ab305220, ab305367, ab305370).
Target Information
CD8 alpha is a transmembrane glycoprotein that functions as a co-receptor for the T-cell receptor (TCR), mainly found on cytotoxic T cells. It plays a crucial role in T cell signaling and cytotoxic activity by binding to MHC class I molecules, aiding the immune response against infected or malignant cells. CD8 alpha is associated with several diseases, particularly those involving immune system dysfunction

Supplementary info

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

Activity summary

CD8 alpha also known as CD8A or CD8 protein is a glycoprotein subunit expressed on the surface of cytotoxic T lymphocytes. It has a mass of approximately 32 kDa. Found on the surface cell membrane CD8 alpha functions primarily in the immune response specifically in the recognition of antigens bound to major histocompatibility complex (MHC) class I molecules. Often scientists use CD8 antibodies for detection and CD8 IHC or immunohistochemistry for localization studies.

Biological function summary

The CD8 alpha protein plays a critical role in T-cell mediated immune responses. It forms a heterodimer with the CD8 beta chain creating the CD8 alpha-beta complex that strengthens T-cell interaction with antigen-presenting cells. CD8 alpha also helps in signaling processes that activate T cells equipping them to destroy infected or malignant cells. Researchers often study CD8 alpha peptides to understand its interactions better.

Pathways

CD8 alpha is integral to the T-cell receptor signaling pathway and the cytotoxic T lymphocyte (CTL) pathway. The T-cell receptor complex which includes the CD8 molecule transmits signals that are important for T-cell activation and function. CD8 interacts with key proteins such as the T-cell receptor (TCR) and MHC class I molecules facilitating targeted responses against pathogens. These pathways highlight CD8 alpha’s role in adaptive immunity.

Associated diseases and disorders

CD8 alpha is most prominently associated with viral infections and cancer. Conditions like HIV and some forms of leukemia show altered CD8 function highlighting the protein's role in immune surveillance. In HIV infection for instance CD8 T cells reduce in number impairing the immune response. CD8 alpha’s connection to the immune system places it alongside other immune proteins such as CD4 and MHC molecules in the context of immune dysfunction.

Product promise

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13 product images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [CAL66] (ab237709)
Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD8 alpha with ab237709 at 1/2000 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 using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
Immunoprecipitation - Anti-CD8 alpha antibody [CAL66] (ab237709)
CD8 alpha was immunoprecipitated from 0.35 mg of human thymus lysate with ab237709 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab237709 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used as secondary antibody at 1/5000 dilution.
Lane 1: Human thymus lysate 10 μg (Input).
Lane 2: ab237709 IP in human thymus lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab237709 in human thymus lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 100 seconds.
All lanes: Immunoprecipitation - Anti-CD8 alpha antibody [CAL66] (ab237709)
Predicted band size: 26 kDa
Observed band size: 26 kDa
Flow Cytometry - Anti-CD8 alpha antibody [CAL66] (ab237709)
Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween 20 permeacblized human PBMC (peripheral blood mononuclear cells) labeling CD8 alpha with ab237709 at 1/500 dilution (Right) compared with a Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Left).
Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (Goat Anti-Rabbit IgG Fc (Alexa Fluor® 488) preadsorbed ab150097), at 1/5000 dilution was used as the secondary antibody.
Cells were surface stained with anti-CD8 alpha conjugated to BV421.The fixed with 2% PFA for 10min followed by intracellularly stained with rabbit IgG (Left) or ab237709 (Right). They recognize same populations.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [CAL66] (ab237709)
Immunohistochemical analysis of paraffin-embedded human colon tissue labeling CD8 alpha with ab237709 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the T lymphocytes in human colon 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 using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [CAL66] (ab237709)
Immunohistochemical analysis of paraffin-embedded human gastric carcinoma tissue labeling CD8 alpha with ab237709 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the T lymphocytes in human gastric carcinoma 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 using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [CAL66] (ab237709)
Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling CD8 alpha with ab237709 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the rat spleen 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 using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
Immunocytochemistry/ Immunofluorescence - Anti-CD8 alpha antibody [CAL66] (ab237709)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized human PBMC (peripheral blood mononuclear cells) labeling CD8 alpha with ab237709 at 1/100 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 subsets of human PBMC. The nuclear counter stain is DAPI (blue). CD3 is detected with an anti-CD3 antibody-Alexa Fluor^® 647 comjugate 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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [CAL66] (ab237709)
Formalin-fixed, paraffin-embedded human tonsil tissue stained for CD8 alpha using ab237709 at 0.25 μg/ml in immunohistochemical analysis.
This image is courtesy of ImmunoAtlas.
Multiplex immunohistochemistry - Anti-CD8 alpha antibody [CAL66] (ab237709)
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-CD8 alpha antibody [CAL66] (ab237709)
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-CD8 alpha antibody [CAL66] (ab237709)
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-CD8 alpha antibody [CAL66] (ab237709)
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-CD8 alpha antibody [CAL66] (ab237709)
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.