Biolegend, 123413, Pacific Blue™ anti-mouse CD21/CD35 (CR2/CR1) Antibody, 25μg

CD21, also known as CR2 (complement receptor 2) and C3d receptor, binds C3d and iC3b. It is also a receptor of Epstein-Barr virus. CD35, also known as CR1, binds C3b, iC3b, C4b, and iC4b. CD21/CD35 is primarily expressed on B lymphocytes, mast cells, follicular dendritic cells, macrophages, and activated granulocytes. CD21/CD35 forms part of the B-cell antigen receptor complex with CD19 and CD81 and is involved in signal transduction.
25μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: CD35/CFA
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography, and conjugated with Pacific Blue™ 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 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 ≤ 1.0 µg per 106 cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application. * Pacific Blue™ has a maximum emission of 455 nm when it is excited at 405 nm. Prior to using Pacific Blue™ conjugate for flow cytometric analysis, please verify your flow cytometer's capability of exciting and detecting the fluorochrome. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Violet Laser (405 nm)
Application Notes: Additional reported application (for relevant formats) include: spatial biology (IBEX)5,6.
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): Boackle S, et al. 2001 Immunity 15:775. de Andres B, et al. 2012. J. Immunol. 189:2300. PubMed Chiu YK, et al. 2014. J Immunol. 193:2207. PubMed Koening PA, et al. 2014. J Biol Chem. 289:34490. 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: Lütge M, et al. 2023. Nat Immunol. . PubMed Voss LF, et al. 2023. Front Immunol. 13:1021370. PubMed Bhattacharya Y 2014. J Exp Med. 211:841. PubMed Farsakoglu Y et al. 2019. Cell reports. 26(9):2307-2315 . PubMed Menzel L, et al. 2021. Cell Rep. 37:109878. PubMed Juul-Madsen K, et al. 2021. Proc Natl Acad Sci U S A. 118:. PubMed Dubey LK, et al. 2019. Cell Rep. 27:2442. PubMed Avery DT, et al. 2018. J Exp Med. 215:2073. PubMed Duan L, et al. 2021. Immunity. 54:2256. PubMed Onodera T, et al. 2016. J Immunol. 196: 4172 - 4184. PubMed Duong B, et al. 2010. J Exp Med. 207:173. PubMed Noviski M, et al. 2018. Elife. 7:e35074. PubMed Lofano G, et al. 2015. J Immunol. 195: 1617-1627. PubMed Simoni L, et al. 2020. Cell Rep. 33:108330. PubMed Seeley-Fallen M, et al. 2014. Proc Natl Acad Sci U S A. 111:9881. PubMed Radice E, et al. 2020. Cell Reports. 32(5):107951. PubMed Gill R, et al. 2017. Toxicol Appl Pharmacol. 330:22. PubMed
RRID: AB_2085159 (BioLegend Cat. No. 123413) AB_2085158 (BioLegend Cat. No. 123414)
Structure: 145 kD/190 kD type I transmembrane glycoprotein
Distribution: B cells, follicular dendritic cells
Function: Signal transduction
Ligand/Receptor: Associated with CD19/CD81. CD21 binds C3d, iC3b, and EBV; CD35 binds C3b, iC3b, C4b, and iC4b.
Cell Type: B cells, Dendritic cells
Biology Area: Cell Biology, Costimulatory Molecules, Immunology, Neuroinflammation, Neuroscience
Molecular Family: Adhesion Molecules, CD Molecules
Antigen References: 1. Kozono Y, et al. 1998. J. Immunol. 160:1562. 2. Shimizu I, et al. 2007. Blood 109:1773. 3. Roozendaal R and MC. Carroll. 2007. Immunol. Rev. 219:157.
Gene ID: 1290212946
UniProt: View information about CD21/CD35 on UniProt.org
Clone: 7E9
Regulatory Status: RUO
Other Names: CR2/CR1
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.