The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/100. PubMed: 18628996
Use a concentration of 0.5 - 2 µg/ml. Predicted molecular weight: 23 kDa.
1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. The recommended starting incubation time is 10min.
Use at an assay dependent concentration. PubMed: 20126467
Use at an assay dependent concentration. PubMed: 23874197
The CD3 complex mediates signal transduction.
Defects 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.
Predicted band size : 23 kDa Observed band size : 23 kDa
Lanes 1 - 6: Merged signal (red and green). Green – ab5690 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 ab5690 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.
No positive immunostaining for CD3ε, a pan T cell marker (ab5690), was detected in the corneas of scrambled siRNA-treated mice (A) at 5 days p.i. In contrast, positive immunostaining (red) was observed in the peripheral cornea of HIF-1α silenced animals (B). The control sections shown in (C) and (D) were immunostained with species-specific IgG and were positive for SYTOX Green nuclear stain only. Images shown are representative of three independent experiments each with three mice per group. Magnification = 180×; inset = 335×.
Immunohistochemistry (Frozen sections) - Anti-CD3 antibody (ab5690)This image is courtesy of an Abreview submitted by Dr Alexandre Garin
ab5690 at 1/100 staining mouse lymph node tissue sections by IHC-Fr. The tissue was acetone fixed and blocked with serum before incubation with the antibody for 1 hour. An Alexa Fluor ® 488 conjugated goat anti-rabbit antibody was used as the secondary.
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 ab5690 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
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 antibody (ab5690)This image is courtesy of an Abreview submitted by Dr Reza Jalili
ab5690 staining rat spleen tissue sections by IHC-P. Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in sodium citrate buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) prior to blocking in 5% normal goat serum + 1% BSA for 2 hours at 37°C. The primary antibody was diluted 1/200 and incubated with the sample for 16 hours at 4°C. A biotinylated goat anti-rabbit antibody, diluted 1/100, was used as the secondary.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 antibody (ab5690)Image courtesy of an anonymous Abreview.
ab5690 staining CD3 in the lymphatic nodule of rat spleen by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer pH 6.0. Samples were then blocked with 1% BSA for 25 minutes at 20°C followed by incubation with the primary antibody at a 1/100 dilution for 1 hour at 20°C. A biotin-conjugated goat anti-rat polyclonal was used undiluted as the secondary antibody. CD3 positive cells are mainly distributed in PALS (T-cell region), while sporadic CD3 positive cells are identified in lymphoid follicle and germinal center (B-cell region).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 antibody (ab5690)This image is courtesy of an Anonymous Abreview.
ab5690 staining CD3 in human lymph node tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded tissue sections). The sections were fixed in paraformaldehyde and subjected to heat-mediated antigen retrieval in citric buffer pH 6.0, prior to blocking with 10% serum for 1 hour at 20°C. The primary antibody was diluted 1/100 and incubated with the sample for 12 hours at 4°C. An HRP-conjugated goat anti-rabbit polyclonal was used as the secondary antibody, diluted 1/200.
Connolly NP et al. Genetically engineered rat gliomas: PDGF-driven tumor initiation and progression in tv-a transgenic rats recreate key features of human brain cancer. PLoS One12:e0174557 (2017).
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