The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. ab172730-Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration. PubMed: 18658050
1/100. 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.
1/200. Predicted molecular weight: 23 kDa.
Use at an assay dependent concentration.
FunctionThe CD3 complex mediates signal transduction.
Involvement in diseaseDefects in CD3D are a cause of severe combined immunodeficiency autosomal recessive T-cell-negative/B-cell-positive/NK-cell-positive (T(-)/B(+)/NK(+) SCID) [MIM:608971]. A form of severe combined immunodeficiency (SCID), a genetically and clinically heterogeneous group of rare congenital disorders characterized by impairment of both humoral and cell-mediated immunity, leukopenia, and low or absent antibody levels. Patients present in infancy recurrent, persistent infections by opportunistic organisms. The common characteristic of all types of SCID is absence of T-cell-mediated cellular immunity due to a defect in T-cell development.
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 Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) 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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 antibody [SP7] (ab16669)This image is courtesy of an Abreview submitted by Dr Mal Niladri
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.
Immunohistochemistry (Frozen sections) - Anti-CD3 antibody [SP7] (ab16669)Image courtesy of an anonymous Abreview.
ab16669 staining CD3 in murine brain tissue by Immunohistochemistry (Frozen sections). Tissue was fixed in acetone, blocked using 5% serum for 30 minutes at 25°C and then incubated with ab16669 at a 1/200 dilution for 18 hours at 4°C. The secondary used was an undiluted HRP conjugated goat anti-rabbit polyclonal.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 antibody [SP7] (ab16669)Image from Moussion C et al., PLoS One. 2008 Oct 6;3(10):e3331. Fig 2.; doi:10.1371/journal.pone.0003331; October 6, 2008, PLoS ONE 3(10): e3331.
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.
Kanazawa H et al. Durable Benefits of Cellular Postconditioning: Long-Term Effects of Allogeneic Cardiosphere-Derived Cells Infused After Reperfusion in Pigs with Acute Myocardial Infarction. J Am Heart Assoc5:N/A (2016).
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