Biolegend, 100240, Alexa Fluor® 594 anti-mouse CD3 Antibody, 100μg

CD3, also known as T3, is a member of the Ig superfamily and primarily expressed on T cells, NK-T cells, and at different levels on thymocytes during T cell differentiation. CD3 is composed of CD3ε, δ, γ and ζ chains. It forms a TCR complex by associating with TCR α/β or γ/δ chains. CD3 plays a critical role in TCR signal transduction, T cell activation, and antigen recognition by binding the peptide/MHC antigen complex
100μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: γδTCR-positive T-T hybridoma D1
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 594 under optimal conditions.
Concentration: 0.5 mg/mL
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: IHC-F - Quality tested FC, 3D IHC - Verified SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by immunohistochemical staining on frozen tissue sections. For immunohistochemistry, a concentration range of 1.0 - 5.0 µg/mL is suggested. For flow cytometric staining, the suggested use of this reagent is ≤ 0.25 µg per million cells in 100 µL volume. For 3D immunohistochemistry on formalin-fixed tissues, a concentration of 5.0 µg/mL is suggested. It is recommended that the reagent be titrated for optimal performance for each application. * Alexa Fluor® 594 has an excitation maximum of 590 nm, and a maximum emission of 617 nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Application Notes: Additional reported application (for relevant formats) include: spatial biology (IBEX)1,2.
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): Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Soon MSF, et al. 2020. Nat Immunol. 1.984027778. PubMed Mueller JPJ, et al. 2023. Front Immunol. 14:1034032. PubMed Backlund C, et al. 2022. Proc Natl Acad Sci U S A. 119:e2204078119. PubMed Baptista AP et al. 2019. Immunity. 50(5):1188-1201 . PubMed Matsumoto R, et al. 2021. Front Pharmacol. 12:715752. PubMed Waide ML, et al. 2020. Cell Rep. 33:108503. PubMed Menzel L, et al. 2021. Cell Rep. 37:109878. PubMed Liu M, et al. 2020. Nature. 587:115. PubMed Wong HS, et al. 2021. Cell. . PubMed Mehta AK, et al. 2021. Nat Cancer. 2:66. PubMed Prados A, et al. 2021. Nat Immunol. 22:510. PubMed Qi S et al. 2016. eLife. 5 pii: e14756. PubMed Wennerberg E, et al. 2022. Sci Transl Med. 14:eabe8195. PubMed Chung YM, et al. 2021. J Immunother Cancer. 9:. PubMed Palacio L, et al. 2019. Aging Cell. 18(4):e12971. PubMed Matryba P, et al. 2020. J Immunol. 1395:204. PubMed Wang X, et al. 2021. EMBO J. 40:e105926. PubMed Pantelidou C, et al. 2022. NPJ Breast Cancer. 8:102. PubMed Lederer K, et al. 2020. Immunity. 53(6):1281-1295.e5. PubMed Lal JC, et al. 2021. Breast Cancer Res. 23:83. PubMed
RRID: AB_2563427 (BioLegend Cat. No. 100240)
Structure: Ig superfamily, CD3/TCR, 20 kD
Distribution: Thymocytes (differentiation dependent), mature T cells, NK-T cells
Function: Antigen recognition, TCR signal transduction, T cell activation
Ligand/Receptor: Peptide antigen/MHC-complex
Antigen References: 1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press. 2. Davis MM. 1990. Annu. Rev. Biochem. 59:475. 3. Weiss A, et al. 1994. Cell 76:263.
Gene ID: 12502
UniProt: View information about CD3 on UniProt.org
Clone: 17A2
Regulatory Status: RUO
Other Names: T cell antigen receptor complex, T3
Isotype: Rat IgG2b, 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.