Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)

Rabbit Recombinant Monoclonal CD3E antibody. Carrier free. Suitable for mIHC, IHC-P, IP, WB, IHC-Fr, Flow Cyt (Intra) and reacts with Mouse, Rat, Human samples. Cited in 1 publication.

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Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (AB251607), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Constituents: PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

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

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	mIHC	IHC-P	IP	WB	ICC/IF	IHC-Fr	Flow Cyt (Intra)
Human	Expected	Tested	Expected	Expected	Not recommended	Expected	Tested
Mouse	Tested	Tested	Tested	Expected	Not recommended	Tested	Tested
Rat	Tested	Tested	Expected	Expected	Not recommended	Expected	Expected

Tested
Tested

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

Expected
Expected

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

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Species Rat	Dilution info -	Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Species Human	Dilution info -	Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes -

Expected
Expected

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

Expected
Expected

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

Not recommended
Not recommended

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

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes -

Expected
Expected

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

Tested
Tested

Species	Dilution info	Notes
Species Mouse, 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 CD3E

Function

Part of the TCR-CD3 complex present on T-lymphocyte cell surface that plays an essential role in adaptive immune response. When antigen presenting cells (APCs) activate T-cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains CD3D, CD3E, CD3G and CD3Z. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways (PubMed:2470098). In addition of this role of signal transduction in T-cell activation, CD3E plays an essential role in correct T-cell development. Initiates the TCR-CD3 complex assembly by forming the two heterodimers CD3D/CD3E and CD3G/CD3E. Participates also in internalization and cell surface down-regulation of TCR-CD3 complexes via endocytosis sequences present in CD3E cytosolic region (PubMed:10384095, PubMed:26507128). In addition to its role as a TCR coreceptor, it serves as a receptor for ITPRIPL1. Ligand recognition inhibits T-cell activation by promoting interaction with NCK1, which prevents CD3E-ZAP70 interaction and blocks the ERK-NFkB signaling cascade and calcium influx (PubMed:38614099).

Alternative names

CD3e, T3E, CD3E, T-cell surface glycoprotein CD3 epsilon chain, T-cell surface antigen T3/Leu-4 epsilon chain

Recommended products

Rabbit Recombinant Monoclonal CD3E antibody. Carrier free. Suitable for mIHC, IHC-P, IP, WB, IHC-Fr, Flow Cyt (Intra) and reacts with Mouse, Rat, Human samples. Cited in 1 publication.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Constituents: PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Carrier free: Yes
Clone number: CAL57
Purification technique: Affinity purification Protein A
Concentration
Purification notes: Purity is greater than 99%.

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: +4°C
Storage information: Do Not Freeze

Notes

ab251607 is the carrier-free version of Anti-CD3 epsilon antibody [CAL57] ab237721.

Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.

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:
Terms & Conditions.

27 product images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on the T lymphocytes in the human tonsil. The section was incubated with Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Immunohistochemical analysis of paraffin-embedded human colon tissue labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on the T lymphocytes in the human colon. The section was incubated with Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Immunohistochemistry (Frozen sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse thymus tissue labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/500 dilution (green), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on T cell surface (PMID: 8490660) is observed. Counterstained with DAPI (blue). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution. Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Flow Cytometry (Intracellular) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized mouse splenocytes labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/500 (right panel) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (left panel). 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.
Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min and intracellularly stained with rabbit IgG (Left) or Anti-CD3 epsilon antibody [CAL57] ab237721 (Right). They recognize the same cell population.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Flow Cytometry (Intracellular) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized human peripheral blood mononuclear cell (PBMC) labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/500 (right panel) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (left panel). Goat anti rabbit IgG (Alexa Fluor^® 488, 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-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min and intracellularly stained with rabbit IgG (Left) or Anti-CD3 epsilon antibody [CAL57] ab237721 (Right). They recognize the same cell population.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Immunoprecipitation - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
CD3 epsilon was immunoprecipitated from 0.35 mg EL4.IL2 (Mouse lymphoma T lymphocyte) whole cell lysate with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/30 dilution. Western blot was performed from the immunoprecipitate using Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used at 1/5000 dilution.
Lane 1: EL4.IL2 whole cell lysate 10 μg (Input).
Lane 2: Anti-CD3 epsilon antibody [CAL57] ab237721 IP in EL4.IL2 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-CD3 epsilon antibody [CAL57] ab237721 in EL4.IL2 whole cell lysate.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 5 seconds.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
All lanes: Immunoprecipitation - Anti-CD3 epsilon antibody [CAL57] (Anti-CD3 epsilon antibody [CAL57] ab237721)
Predicted band size: 23 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on the T lymphocytes in the rat spleen. The section was incubated with Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD3 epsilon with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on the T lymphocytes in the mouse spleen. The section was incubated with Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
Formalin-fixed, paraffin-embedded mouse spleen tissue stained for CD3 epsilon using Anti-CD3 epsilon antibody [CAL57] ab237721 at 0.5 μg/ml in immunohistochemical analysis.
This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse lymph node staining of NCR1 with Anti-NCR1 antibody [EPR23097-35] ab233558 at a 1/500 (1.062 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-NCR1 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse lymph node.
Panel B: anti-NCR1 staining natural killer cells in mouse lymph node.
Panel C: anti-CD19 staining B lymphocytes in mouse lymph node..
Panel D: anti-CD3 staining T lymphocytes in mouse lymph node.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-NCR1 antibody [EPR23097-35] ab233558, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen staining of NCR1 with Anti-NCR1 antibody [EPR23097-35] ab233558 at a 1/500 (1.062 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-NCR1 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse spleen.
Panel B: Anti-NCR1 staining natural killer cells in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-NCR1 antibody [EPR23097-35] ab233558, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen staining of F4/80 with Anti-F4/80 antibody [EPR26545-166] ab300421 at a 1/5000 (0.1 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-F4/80 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse spleen.
Panel B: anti-F4/80 staining macrophages in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-F4/80 antibody [EPR26545-166] ab300421, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse lymph node staining of F4/80 with Anti-F4/80 antibody [EPR26545-166] ab300421 at a 1/5000 (0.1 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-F4/80 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse lymph node.
Panel B: anti-F4/80 staining macrophages in mouse lymph node.
Panel C: anti-CD19 staining B lymphocytes in mouse lymph node.
Panel D: anti-CD3 staining T lymphocytes in mouse lymph node.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-F4/80 antibody [EPR26545-166] ab300421, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Immunohistochemistry analysis of (Formalin/PFA-fixed paraffin-embedded sections) Rat lymph node tissue labelling MAdCAM1 with Anti-MAdCAM1 antibody [EPR27223-58] ab309487 at 1:100 dilution (B), CD3 epsilon with ab237721at 1:2000 dilution (C) and CD20 with Anti-CD20 antibody [SP32] ab64088 at 1:100 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Panel A: merged staining of anti-MAdCAM (green; Opal™520), anti-CD3 (gray; Opal™690) and anti-CD20 (magenta; Opal™570) on rat lymph node.
Panel B: anti-MAdCAM staining high endothelial venules in rat lymph node.
Panel C: ant-CD3 staining B lymphocytes in rat lymph node.
Panel D: ant-CD20 staining B lymphocytes in rat lymph node.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-MAdCAM1 antibody [EPR27223-58] ab309487, Anti-CD3 epsilon antibody [CAL57] ab237721 and Anti-CD20 antibody [SP32] ab64088 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation ( Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen tissue staining Neutrophil Elastase with Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335 at a 1/500 ( 1.076 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at a 1/1000 ( 0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at a 1/2000 ( 0.264 µg/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-Neutrophil Elastase (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™690) on mouse spleen.Panel B: anti-Neutrophil Elastase staining neutrophils in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation ( Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse thymus tissue staining Neutrophil Elastase with Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335 at a 1/500 ( 1.076 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at a 1/1000 ( 0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at a 1/2000 ( 0.264 µg/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-Neutrophil Elastase (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™690) on mouse spleen.Panel B: anti-Neutrophil Elastase staining neutrophils in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation ( Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse bone marrow tissue staining Neutrophil Elastase with Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335 at a 1/500 ( 1.076 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at a 1/1000 ( 0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at a 1/2000 ( 0.264 µg/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-Neutrophil Elastase (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™690) on mouse spleen.Panel B: anti-Neutrophil Elastase staining neutrophils in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation ( Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse lymph node tissue staining Neutrophil Elastase with Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335 at a 1/500 ( 1.076 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at a 1/1000 ( 0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at a 1/2000 ( 0.264 µg/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-Neutrophil Elastase (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™690) on mouse spleen.Panel B: anti-Neutrophil Elastase staining neutrophils in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse thymus staining of F4/80 with Anti-F4/80 antibody [EPR26545-166] ab300421 at a 1/5000 (0.1 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-F4/80 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse thymus.
Panel B: anti-F4/80 staining macrophages in mouse thymus.
Panel C: anti-CD19 staining B lymphocytes in mouse thymus.
Panel D: anti-CD3 staining T lymphocytes in mouse thymus.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-F4/80 antibody [EPR26545-166] ab300421, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Immunohistochemistry analysis of (Formalin/PFA-fixed paraffin-embedded sections) Mouse lymph node tissue labelling MAdCAM1 with Anti-MAdCAM1 antibody [EPR27223-58] ab309487 at 1:100 dilution (B), CD3 epsilon with ab237721at 1:2000 dilution (C) and CD20 with Anti-CD20 antibody [SP32] ab64088 at 1:100 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Panel A: merged staining of anti-MAdCAM (green; Opal™520), anti-CD3 (gray; Opal™690) and anti-CD20 (magenta; Opal™570) on mouse lymph node.
Panel B: anti-MAdCAM staining high endothelial venules in mouse lymph node.
Panel C: ant-CD3 staining B lymphocytes in mouse lymph node.
Panel D: ant-CD20 staining B lymphocytes in mouse lymph node.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-MAdCAM1 antibody [EPR27223-58] ab309487, Anti-CD3 epsilon antibody [CAL57] ab237721 and Anti-CD20 antibody [SP32] ab64088 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse small intestine tissue staining CD68 with Anti-CD68 antibody [EPR23917-164] ab283654 at a 1/100 dilution, Anti-CD19 antibody [EPR23174-145] ab245235 anti-CD19 used at 1/1000 dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Panel A: merged staining of anti-CD68 (magenta; Opal^™520), anti-CD19 (green; Opal^™690) and anti-CD3 (grey; Opal^™570) on mouse small intestine.

Panel B: anti-CD68 staining macrophages in mouse small intestine.

Panel C: anti-CD19 staining B lymphocytes in mouse small intestine.

Panel D: anti-CD3 staining T lymphocytes in mouse small intestine.

Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-CD68 antibody [EPR23917-164] ab283654, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse colon tissue staining CD68 with Anti-CD68 antibody [EPR23917-164] ab283654 at a 1/100 dilution, Anti-CD19 antibody [EPR23174-145] ab245235 anti-CD19 used at 1/1000 dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Panel A: merged staining of anti-CD68 (magenta; Opal^™520), anti-CD19 (green; Opal^™690) and anti-CD3 (grey; Opal^™570) on mouse colon.

Panel B: anti-CD68 staining macrophages in mouse colon.

Panel C: anti-CD19 staining B lymphocytes in mouse colon.

Panel D: anti-CD3 staining T lymphocytes in mouse colon.

Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-CD68 antibody [EPR23917-164] ab283654, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation (Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining CD23 with Anti-CD23 antibody [EPR28712-26] ab315289 at a 1/2000 dilution (0.253 µg/ml), CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 dilution (0.444 µg/ml), and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/8000 dilution (0.07 µg/ml), followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-CD23 (green; Opal™520), anti-CD19 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on mouse spleen.
Panel B: anti-CD23 staining B lymphocytes in mouse spleen.
Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-CD23 antibody [EPR28712-26] ab315289, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation (Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse lymph node tissue staining CD23 with Anti-CD23 antibody [EPR28712-26] ab315289 at a 1/2000 dilution (0.253 µg/ml), CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 dilution (0.444 µg/ml), and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/8000 dilution (0.07 µg/ml), followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-CD23 (green; Opal™520), anti-CD19 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on mouse lymph node.
Panel B: anti-CD23 staining B lymphocytes in mouse lymph node.
Panel C: anti-CD19 staining B lymphocytes in mouse lymph node.
Panel D: anti-CD3 staining T lymphocytes in mouse lymph node.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-CD23 antibody [EPR28712-26] ab315289, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using the same antibody clone in a different buffer formulation (Anti-CD3 epsilon antibody [CAL57] ab237721).
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse thymus tissue staining CD23 with Anti-CD23 antibody [EPR28712-26] ab315289 at a 1/2000 dilution (0.253 µg/ml), CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 dilution (0.444 µg/ml), and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/8000 dilution (0.07 µg/ml), followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A:merged staining of anti-CD23 (green; Opal™520), anti-CD19 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on mouse thymus.
Panel B: anti-CD23 staining B lymphocytes in mouse thymus.
Panel C: anti-CD19 staining B lymphocytes in mouse thymus.
Panel D: anti-CD3 staining T lymphocytes in mouse thymus.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-CD23 antibody [EPR28712-26] ab315289, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse bone marrow staining of F4/80 with Anti-F4/80 antibody [EPR26545-166] ab300421 at a 1/5000 (0.1 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-F4/80 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse bone marrow.
Panel B: anti-F4/80 staining macrophages in mouse bone marrow.
Panel C: anti-CD19 staining B lymphocytes in mouse bone marrow.
Panel D: anti-CD3 staining T lymphocytes in mouse bone marrow.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-F4/80 antibody [EPR26545-166] ab300421, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.
Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [CAL57] - BSA and Azide free (ab251607)
This data was developed using Anti-CD3 epsilon antibody [CAL57] ab237721, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse thymus staining of NCR1 with Anti-NCR1 antibody [EPR23097-35] ab233558 at a 1/500 (1.062 µg/ml) dilution, CD19 with Anti-CD19 antibody [EPR23174-145] ab245235 at 1/1000 (0.444 µg/ml) dilution and CD3 with Anti-CD3 epsilon antibody [CAL57] ab237721 at 1/2000 ( 0.264 µg/ml) dilution.
Panel A: merged staining of anti-NCR1 (magenta; Opal™690), anti-CD19 (green; Opal™520) and anti-CD3 (gray; Opal™570) on mouse thymus.
Panel B: anti-NCR1 staining natural killer cells in mouse thymus.
Panel C: anti-CD19 staining B lymphocytes in mouse thymus.
Panel D: anti-CD3 staining T lymphocytes in mouse thymus.
Nuclear DNA was labelled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-NCR1 antibody [EPR23097-35] ab233558, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.