Anti-CD3 antibody [SP7] - BSA and Azide free (ab205228)
Key features and details
- Rabbit monoclonal [SP7] to CD3 - BSA and Azide free
- Suitable for: Flow Cyt (Intra), WB, IHC-P, mIHC
- Reacts with: Mouse, Rat, Human
- Isotype: IgG
Related conjugates and formulations
Overview
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Product name
Anti-CD3 antibody [SP7] - BSA and Azide free
See all CD3 primary antibodies -
Description
Rabbit monoclonal [SP7] to CD3 - BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, mIHCmore details -
Species reactivity
Reacts with: Mouse, Rat, Human
Predicted to work with: Sheep, Rabbit, Horse, Chicken, Cow, Cat, Dog, Pig, Baboon, Woodchuck -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Recombinant Human CD3 epsilon protein (ab114153), Jurkat whole cell lysate. Human, mouse and rat thymus tissue lysate. IHC-P: Human tonsil tissue. Mouse epidydimal fat pad and lymph node tissues. Rat infarcted heart and spleen tissues. Flow Cyt (intra): Human peripheral blood lymphocytes. Jurkat cells. mIHC: Human tonsil tissue, human duodenum tissue, human colon tissue. Mouse and rat spleen tissue.
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General notes
This is the BSA and azide free formulation of ab16669.
This antibody is suitable for staining normal and neoplastic T cells in formalin-fixed, paraffin-embedded tissues.
This product is FOR RESEARCH USE ONLY. For commercial use, please contact partnerships@abcam.com.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at +4°C. Do Not Freeze. -
Storage buffer
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Tissue culture supernatant -
Clonality
Monoclonal -
Clone number
SP7 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab205228 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) | ||
WB |
Use at an assay dependent concentration. Predicted molecular weight: 23 kDa.
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IHC-P | (1) |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min. |
mIHC |
Use at an assay dependent concentration.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) |
Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 23 kDa. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min. |
mIHC
Use at an assay dependent concentration. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) |
Images
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Panel A: merged staining of anti-CD68 (gray; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human tonsil. Secondary antibody was Opal Polymer HRP Ms + Rb, and counterstaining was with DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD68 stained on macrophages with ab213363 1/500 dilutionThe section was incubated in three rounds of staining: in the order of ab213363 and ab16669 for 30 mins, then ab237772 for 10 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.
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.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Panel A: merged staining of anti-CD68 (magenta; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human spleen. Secondary antibody was Opal Polymer HRP Ms + Rb, and counterstaining was with DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD68 stained on macrophages with ab213363 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab213363 and ab16669 for 30 mins, then ab237772 for 10 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.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.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Panel A: merged staining of anti-CD31 (gray; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human tonsil. Secondary antibody was Opal Polymer HRP Ms + Rb, nuclear counterstain was DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD31 stained on endothelial cells and immune cell subsets with ab207090 at 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab207090 and ab16669 for 30 mins, then ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins -
This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Panel A: merged staining of anti-CD31 (gray; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human spleen. Secondary antibody was Opal Polymer HRP Ms + Rb, nuclear counterstain was DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD31 stained on endothelial cells with ab207090 at 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab207090 and ab16669 for 30 mins, then ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins -
This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human thymus tissue labeling CD3 with ab16669 at 1/500 dilution, LY75/DEC-205 with ab208649 at 1/15000, and CD68 with ab213363 at 1/500 dilution.
Panel A: merged staining of anti-CD68 (magenta; Opal™690), anti-CD3 (green; Opal™520) and anti-LY75/DEC-205 (red; Opal™570) on human thymus.
Panel B: anti-CD3 stained on T cells.
Panel C: anti-LY75/DEC-205 stained on thymic cortical epithelium and dendritic cells.
Panel D: anti-CD68 stained on macrophages.
Sections were treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins before antibody incubation. The section was incubated in three rounds of staining: in the order of ab213363, ab16669, and ab208649 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.DAPI was used as a nuclear counterstain.
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. -
This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat spleen tissue labeling Sialoadhesin/CD169, CD3 and CD20 with ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and ab236434 at 1:5000 dilution.
Panel A: merged staining of anti-Sialoadhesin/CD169 (red; Opal™690), anti-CD3 (green; Opal™520) and anti-CD20 (gray; Opal™570) on rat spleen.
Panel B: anti-Sialoadhesin/CD169 stained on macrophages.
Panel C: anti-CD3 stained on T cells.
Panel D: anti-CD20 stained on B cells.The section was incubated in three rounds of staining: in the order of ab312840, ab16669, and ab236434 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.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen tissue labeling Sialoadhesin/CD169, CD3 and CD20 with ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and ab236434 at 1:5000 dilution.
Panel A: merged staining of anti-Sialoadhesin/CD169 (red; Opal™690), anti-CD3 (green; Opal™520) and anti-CD20 (gray; Opal™570) on mouse spleen.
Panel B: anti-Sialoadhesin/CD169 stained on macrophages.
Panel C: anti-CD3 stained on T cells.
Panel D: anti-CD20 stained on B cells.The section was incubated in three rounds of staining: in the order of ab312840, ab16669, and ab236434 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.
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Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (ab240401, gray; Opal™690), anti-CD3 (ab16669, green; Opal™520) and anti-CD68 (ab213363, red; Opal™570) on human colon. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-CD3 stained on T cells. Panel D: anti-CD68 stained on macrophages. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab240401 (1/8000 dilution), ab16669 (1/150 dilution), and ab213363 (1/500 dilution) 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. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using ab16669, the same antibody clone in a different buffer formulation.
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Fluorescence multiplex immunohistochemical analysis of the human duodenum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (ab240401, gray; Opal™690), anti-CD3 (ab16669, green; Opal™520) and anti-CD68 (ab213363, red; Opal™570) on human duodenum. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-CD3 stained on T cells. Panel D: anti-CD68 stained on macrophages. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab240401 (1/8000 dilution), ab16669 (1/150 dilution), and ab213363 (1/500 dilution) 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. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using ab16669, the same antibody clone in a different buffer formulation.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
IHC image of CD3 staining in a formalin fixed, paraffin embedded normal rat spleen tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
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All lanes : Anti-CD3 epsilon antibody [SP7] (ab16669) at 1/25 dilution
Lane 1 : THP1 whole cell lysate (-ve control)
Lane 2 : Raji whole cell lysate (-ve control)
Lane 3 : Jurkat whole cell lysate
Lane 4 : Human Thymus tissue lysate
Lane 5 : Mouse Thymus tissue lysate
Lane 6 : Rat Thymus tissue lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Developed using the ECL technique.
Predicted band size: 23 kDa
Observed band size: 23 kDaThis data was developed using ab16669, the same antibody clone in a different buffer formulation.
Lanes 1 - 6: Merged signal (red and green). Green – ab16669 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab16669 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).
Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences®).
The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.
Sodium citrate antigen retrieval (Leica ER1, pH6.0, 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.
DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Human peripheral blood lymphocytes stained with ab16669 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab16669, 1/1000 dilution) for 30 min at 4°C. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
IHC image of CD3 staining in a formalin fixed, paraffin embedded normal human tonsil tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
IHC image of CD3 staining in mouse lymph node formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of Formaldehyde fixed, paraffin-embedded rat spleen tissue sections labelling CD3 with ab16669 at a dilution of 1/100. Biotin conjugated Goat Anti-Rabbit IgG at 1/300 dilution was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
IHC using multi-spectral imaging on human lymph node (A-C) obtained from men with mCRPC. A) H+E staining and B) single-color images (plus nuclear stain; DAPI) of CD3 (ab16669), CD8 (ab101500), CD163, PD-L1, cytokeratin (CK), DAPI and C) merged. H+E stating at 20X magnification; multi-spectral images 200X magnification.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
ab16669 staining CD3 in Mouse Epidydimal fat pad tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue sections were fixed with formaldehyde, blocked with 5% serum for 4 hours at 25°C and permeabilized with Triton X-100. Samples were incubated with primary antibody (1/100 in PBST with BSA and goat serum) for 4°C at 12 hours. An Alexa Fluor® 568 goat anti-rabbit IgG (H + L) cross adsorbed was used as the secondary antibody.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
ab16669 staining rat infarcted heart tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
Myocardial infarction was produced in a rat model following the ligation of the left anterior descending (LAD) coronary artery. Tissue was harvested 6 w following infarct, fixed with Histochoice for 72 hr, paraffin sectioned and the slide was then baked prior to CD3 staining. ab16669 at 1/200 was incubated overnight at 4°C. The image was taken with a confocal laser scanning microscope and shows cells giving strong inmmunofluorescence staining for CD3 antigen (green), indicating presence of cells of T-lymphocytes origin in the infarct zone of the heart tissue, counterstained nuclei with DAPI (blue). Note, CD3 tended to be present in nests of 2-5 cells that were non-uniformly distributed in the infarct zone. In addition, the image shows that the CD3 localization is predominantly membrane based and to a certain extent intracytoplasmic.
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This data was developed using ab16669, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of Human tonsil tissue, staining CD3 (green) with ab16669.
Antigen retrieval was performed by heat mediation in citrate buffer (pH 6) and blocked with 5% goat serum and 5% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/100) overnight at 4°C. A Cy3®-conjugated anti-rabbit IgG was used as the secondary antibody.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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Datasheet download
References (11)
ab205228 has been referenced in 11 publications.
- Xu Q et al. Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children. Nat Immunol 24:186-199 (2023). PubMed: 36536106
- Pillon A et al. Actinic keratosis modelling in mice: A translational study. PLoS One 12:e0179991 (2017). PubMed: 28662116
- Linch SN et al. Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice. Proc Natl Acad Sci U S A 113:E319-27 (2016). PubMed: 26729864
- Huang Y et al. Evidence of an oncogenic role of aberrant TOX activation in cutaneous T-cell lymphoma. Blood 125:1435-43 (2015). PubMed: 25548321
- Lastrucci C et al. Molecular and cellular profiles of the resolution phase in a damage-associated molecular pattern (DAMP)-mediated peritonitis model and revelation of leukocyte persistence in peritoneal tissues. FASEB J 29:1914-29 (2015). PubMed: 25609430