Name: Anti-EpCAM antibody [EPR20532-225]
SKU: ab223582
Rating: 5 (2 reviews)

Anti-EpCAM antibody [EPR20532-225] (ab223582) is a rabbit monoclonal antibody detecting EpCAM in Western Blot, Flow Cytometry, IP, IHC-P, ICC/IF, mIHC. Suitable for Human.

- KO validated for confirmed specificity

- Multiplex IHC validated on the Leica BOND® MAX using Opal reagents

- Biophysical QC for unrivalled batch-batch consistency

- Over 20 publications

Related conjugates and formulations

ab237397
Conjugated
PE Anti-EpCAM antibody [EPR20532-225]
ab237398
Conjugated
APC Anti-EpCAM antibody [EPR20532-225]
ab286811
Conjugated
Alexa Fluor® 594 Anti-EpCAM antibody [EPR20532-225]
ab225894
Carrier free
Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free
ab237396
Conjugated
Alexa Fluor® 647 Anti-EpCAM antibody [EPR20532-225]
ab237395
Conjugated
Alexa Fluor® 488 Anti-EpCAM antibody [EPR20532-225]
ab314940
Conjugated
Biotin Anti-EpCAM antibody [EPR20532-225]

Images

Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] (AB223582), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

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

Abcam Recommends

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ICC/IF	IP	Flow Cyt	WB	mIHC	IHC-P
Human	Tested	Tested	Tested	Tested	Tested	Tested

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500 - 1/5000	Notes 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 1/30	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes -

Tested
Tested

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

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	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.

Associated Products

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Target data

See full target information EPCAM

Function

May act as a physical homophilic interaction molecule between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) at the mucosal epithelium for providing immunological barrier as a first line of defense against mucosal infection. Plays a role in embryonic stem cells proliferation and differentiation. Up-regulates the expression of FABP5, MYC and cyclins A and E.

Alternative names

CD326, GA733-2, M1S2, M4S1, MIC18, TACSTD1, TROP1, EPCAM, Epithelial cell adhesion molecule, Ep-CAM, Adenocarcinoma-associated antigen, Cell surface glycoprotein Trop-1, Epithelial cell surface antigen, Epithelial glycoprotein, Epithelial glycoprotein 314, KS 1/4 antigen, KSA, Major gastrointestinal tumor-associated protein GA733-2, Tumor-associated calcium signal transducer 1, EGP, EGP314, hEGP314

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR20532-225
Purification technique: Affinity purification Protein A
Concentration

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-EpCAM antibody [EPR20532-225] (ab223582) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt, ICC/IF, IHC-P, IP, WB, mIHC in human samples.
Anti-EpCAM antibody [EPR20532-225] (ab223582) specifically detects EpCAM (UniProt ID: P16422; Molecular weight: 33kDa) 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-EpCAM antibody [EPR20532-225] (ab223582) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.
The specificity of Anti-EpCAM antibody [EPR20532-225] (ab223582) has been confirmed by testing in knockout samples.
Anti-EpCAM antibody [EPR20532-225] (ab223582) has been cited over 27 times in peer reviewed journals and is trusted by the scientific community.

Related Products
Conjugation-ready, carrier free format available for antibody clone EPR20532-225 - ab225894.
Antibody clone EPR20532-225 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 488, Alexa Fluor® 647, PE, APC, Alexa Fluor® 594, Biotin, Alexa Fluor® 647 (ab237395, ab237396, ab237397, ab237398, ab286811, ab314940).

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

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

Activity summary

EpCAM also known as epithelial cell adhesion molecule is a transmembrane glycoprotein with a molecular weight of around 40 kDa. Alternative names include CD326 and TACSTD1. This protein is expressed on the surface of most epithelial cells and plays a role in cell adhesion. It is involved in maintaining the integrity of epithelial tissues by mediating homotypic cell-cell interactions which are important for the structure and function of tissues.

Biological function summary

EpCAM facilitates signaling pathways that impact cellular processes like proliferation and differentiation. It is not a part of a larger protein complex but interacts directly with other cellular machinery to transmit intracellular signals. EpCAM influences stem cell behavior and is important in tissue homeostasis assisting in both normal regenerative processes and wound healing.

Pathways

EpCAM participates in regulatory mechanisms involving the Wnt/β-catenin signaling pathway which affects gene expression and cell fate decisions. Through its action EpCAM modulates components like E-cadherin which is important for cell adhesion and the maintenance of the epithelial layer. It indirectly influences pathways relating to MYC a transcription factor that regulates cell cycle progression and apoptosis.

Associated diseases and disorders

EpCAM is associated with various types of cancer including colorectal and breast cancer. Its overexpression often correlates with aggressive cancer phenotypes and poor prognosis. Besides cancers EpCAM mutations can lead to congenital tufting enteropathy a rare condition affecting intestinal epithelial structure and function. Aberrant EpCAM expression affects β-catenin levels impacting cellular pathways linked to tumor progression and epithelial integrity.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

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:
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18 product images

Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] (ab223582)
ab223582 staining EpCAM in wild-type A431 cells (top panel) and EpCAM knockout A431 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-Tween 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 with ab223582 at 0.1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.
Flow Cytometry - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Flow cytometry overlay histogram showing wild-type A431 (green line) and EPCAM knockout A431 cells stained with ab223582 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab223582) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was Rabbit IgG (monoclonal) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) used at the same concentration and conditions as the primary antibody (wild-type A431 - black line; EPCAM knockout A431 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
Western blot - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Lanes 1 - 3: Merged signal (red and green). Green - ab223582 observed at 40 kDa. Red - loading control, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab223582 was shown to react with EpCAM in A431 wild-type cells in Western blot. Loss of signal was observed when EpCAM knockout sample was used. A431 wild-type and EpCAM knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab223582 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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-EpCAM antibody [EPR20532-225] (ab223582) at 1/1000 dilution
Lane 1: Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2: EPCAM knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2: Western blot - Human EPCAM knockout A-431 cell line (Human EPCAM knockout A-431 cell line ab261902)
Lane 3: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 35 kDa
Observed band size: 40 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Immunohistochemical analysis of paraffin-embedded human thyroid cancer tissue labeling EpCAM with ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human thyroid cancer is observed (PMID: 15637741). 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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Western blot - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-EpCAM antibody [EPR20532-225] (ab223582) at 1/5000 dilution
Lane 1: HCT 116 (human colorectal carcinoma cell line) lysate at 20 µg
Lane 2: HT-29 (human colorectal adenocarcinoma cell line) 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: 35 kDa
Observed band size: 42 kDa
Exposure time: 3min
Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] (ab223582)
ab223582 staining EpCAM in T47D positive cells (top panel) and HeLa negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-Tween 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 with ab223582 at 0.1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.
Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Immunofluorescent analysis of 4% paraformaledehyde-fixed, 0.1% Triton X-100 permeabilized HT-29 (human colorectal adenocarcinoma cell line) cells labeling EpCAM with ab223582 at 1/500 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 on HT-29 cells.
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) (red) at 1/200 dilution.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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-EpCAM antibody [EPR20532-225] (ab223582)
Immunohistochemical analysis of paraffin-embedded human colon tissue labeling EpCAM with ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human colon is observed (PMID: 15637741). 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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunoprecipitation - Anti-EpCAM antibody [EPR20532-225] (ab223582)
EpCAM was immunoprecipitated from 0.35 mg of HCT 116 (human colorectal carcinoma cell line) whole cell lysate with ab223582 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223582 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10000 dilution.
Lane 1: HCT 116 lysate 10 μg (Input).
Lane 2: ab223582 IP in HCT 116 lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab223582 in HCT 116 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.
All lanes: Immunoprecipitation - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Predicted band size: 35 kDa
Observed band size: 42 kDa
Exposure time: 30s
Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] (ab223582)
ab223582 staining EpCAM in wild-type A431 cells (top panel) and EpCAM knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% PBS-Tween 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 with ab223582 at 1/5000 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Western blot - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-EpCAM antibody [EPR20532-225] (ab223582) at 1/1000 dilution
Lane 1: Human breat cancer lysate at 10 µg
Lane 2: Human fetal kidney lysate at 10 µg
Secondary
Lane 1: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution
Lane 2: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/4000 dilution
Predicted band size: 35 kDa
Observed band size: 42 kDa
Exposure time: 3min
This image is courtesy of ImmunoAtlas.
Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] (ab223582)
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.
Flow Cytometry - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Flow cytometric analysis of HCT 116 (human colorectal carcinoma cell line) cell line labeling EpCAM with ab223582 at 1/500 (red) compared with a rabbit monoclonal IgG Isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), at 1/2000 dilution was used as the secondary antibody.
Total viable cells were gated for the final data.
This image is courtesy of ImmunoAtlas.
Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] (ab223582)
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-EpCAM antibody [EPR20532-225] (ab223582)
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), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048).
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.
Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] (ab223582)
Fluorescence multiplex immunohistochemical analysis of the human endometrium (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-EpCAM (ab223582, magenta; Opal™690), anti-NKG2A (Anti-NKG2A antibody [EPR23737-127] ab260035, green; Opal™520) and anti-CD31 (Anti-CD31 antibody [EPR17259] ab182981, red; Opal™570) on human endometrium. Panel B: anti-NKG2A stained on NK cells. Panel C: anti-CD31 stained on endothelial cells. Panel D: anti-EpCAM stained on glandular cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab223582 at 1/500 dilution (1.008 μg/ml), Anti-NKG2A antibody [EPR23737-127] ab260035 at 1/2000 dilution (0.262 μg/ml) and Anti-CD31 antibody [EPR17259] ab182981 at 1/4000 dilution (0.137 μg/ml) for 30 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. DAPI (blue) was used as a nuclear counter stain.
This image is courtesy of ImmunoAtlas.
Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] (ab223582)
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-EpCAM antibody [EPR20532-225] (ab223582)
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.