Biolegend, 351725, Alexa Fluor® 647 anti-human TCR Vα7.2 Antibody, 25tests

The 3C10 antibody recognizes the Vα7.2 T cell antigen receptor (TCR) α-chain segment which, joined with the Jα33 segment, constitutes an invariant TCR that is a characteristic of the mucosal-associated invariant T cells (MAIT cells). MAIT cells are restricted by a nonpolymorphic class Ib major histocompatibility complex (MHC) molecule, MHC-related molecule 1 (MR1). MAIT cells are present in human blood (1-8% of T cells), mesenteric lymph nodes, liver, and intestinal mucosa.  MAIT cells play a role in detecting and fighting off microbial infections.
25tests
Verified Reactivity: Human
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: Recombinant TCR
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA)
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration: Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: FC - Quality tested SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Red Laser (633 nm)
Application Notes: Associated with an anti-CD161 or -IL18Ra staining, the 3C10 antibody allows unequivocal identification of MAIT cells. Importantly, the Va7.2 segment can also be used by conventional T cells. Therefore, the 3C10 also stains a subset of conventional CD4 and CD8 T cells. Additional reported applications (for the relevant formats) include: spatial biology (IBEX)3,4.
Additional Product Notes: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
Application References(PubMed link indicates BioLegend citation): Martin E, et al. 2009. PLoS Biol. 7:525. Wakao H, et al. 2013. Cell Stem Cell 12:1. PubMed Radtke AJ, et al. 2020. Proc Natl Acad Sci USA. 117:33455-33465. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Qu J, et al. 2024. J Exp Clin Cancer Res. 134:43. PubMed
RRID: AB_2566334 (BioLegend Cat. No. 351725) AB_2566335 (BioLegend Cat. No. 351726)
Structure: Invariant T cell antigen receptor (TCR) α-chain (iTCRa), composed of the invariant α-chain variable region 7.2 (iVα7.2) segment in humans
Distribution: Mucosal-associated invariant T (MAIT) cells and conventional T cells
Cell Type: T cells
Biology Area: Adaptive Immunity, Immunology
Molecular Family: TCRs
Antigen References: 1. Le Bourhis L, et al. 2010. Nat. Immunol. 11:701.
Gene ID: 28692
UniProt: View information about TCR Valpha7.2 on UniProt.org
Clone: 3C10
Regulatory Status: RUO
Other Names: T cell antigen receptor Vα7.2, TRAV1-2, T cell receptor alpha variable 1-2
Isotype: Mouse IgG1, κ
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.