Biolegend, 101611, Alexa Fluor® 647 anti-mouse CD23 Antibody, 25μg

CD23 is a 45 kD protein also known as low affinity IgE Fc receptor, FcεRII, BLAST-2, Ly-42, or B6. It is a member of the Ig family, expressed on conventional B (but not B-1) cells and follicular dendritic cells. CD23 responds to high levels of IgE by downregulating IgE secretion.
25μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: Complex of IgE with Fcε receptor isolated from the mouse B hybridoma cell line O1.2B2
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® 647 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: FC - Quality tested IHC-F - Verified 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 ≤ 0.25 µg per million cells in 100 µl volume. For immunohistochemistry on frozen tissue sections, a concentration range of 2.5 - 10 µg/ml is suggested. It is recommended that the reagent be titrated for optimal performance for each application. * 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: The B3B4 antibody is useful for blocking IgE activity in vivo. Additional reported applications (for the relevant formats) include: immunoprecipitation1, immunofluorescence microscopy, in vitro and in vivo blocking of ligand binding2-4, immunohistochemical staining of acetone-fixed frozen sections5, and spatial biology (IBEX)7,8.
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): Waldschmidt TJ, et al. 1988. J. Immunol. 140:2148. (IP) Rao M, et al. 1987. J. Immunol. 138:1845. (Block) Oshiba A, et al. 1997. J. Immunol. 159:4056. (Block) Dasic G, et al. 1999. Eur. J. Immunol. 29:2957. (Block) Maeda K, et al. 1992. J. Immunol. 148:2340. (IHC) Craig VJ, et al. 2011. Cancer Res. 71:3616. PubMed 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: Lino AC et al. 2018. Immunity. 49(1):120-133 . PubMed Duan L, et al. 2021. Immunity. 54:2256. PubMed Noviski M, et al. 2018. Elife. 7:e35074. PubMed Shiozawa S, et al. 2022. iScience. 25:103537. PubMed
RRID: AB_493479 (BioLegend Cat. No. 101611) AB_2103038 (BioLegend Cat. No. 101612)
Structure: C-lectin family, type II transmembrane glycoprotein, 45 kD
Distribution: B cells, follicular dendritic cells
Function: Regulates B cell activation
Ligand/Receptor: IgE, CD19/CD21/CD81
Cell Type: B cells, Dendritic cells
Biology Area: Immunology
Molecular Family: CD Molecules, Fc Receptors
Antigen References: 1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press. 2. Delespesse G, et al. 1992. Immunol. Rev. 125:77. 3. Flores-Romo L, et al. 1993. Science 261:1038.
Gene ID: 14128
UniProt: View information about CD23 on UniProt.org
Clone: B3B4
Regulatory Status: RUO
Other Names: FcεRII, IgE Fc Receptor, BLAST-2, Ly-42, B6
Isotype: Rat IgG2a, 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.