Biolegend, 405717, Alexa Fluor® 488 anti-mouse IgD Antibody, 25μg

Surface IgD is an important B cell differentiation marker.
25μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
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® 488 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 ≤0.5 µg per million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application. * Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Blue Laser (488 nm)
Application Notes: The 11-26c.2a antibody reacts with immunoglobulin D in all tested mouse haplotypes. The antibody binds membrane IgD expressed on most B cells. The 11-26c.2a antibody neither induces proliferation of splenic B cells nor induces B cell activation. Additional reported applications (for the relevant formats) include: immunohistochemical staining of acetone-fixed frozen sections2,3, and spatial biology (IBEX)10,11.
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): Nitschke L, et al. 1993. P. Natl. Acad. Sci. USA 90:1887. (FC) Weih D, et al. 2001. J. Immunol. 167:1909. (IHC) Koni PA, et al. 2001. J. Exp. Med. 193:741. (IHC) Ahuja A, et al. 2007. J. Immunol. 179:3351. (FC) PubMed Haynes NM, et al. 2007. J. Immunol. 179:5099. (FC) Good-Jacobson KL, et al. 2010. Nat. Immunol. 11:535. (FC) PubMed Tomayko MM, et al. 2010. J. Immunol. 185:7146. PubMed Park SY, et al. 2013. J. Immunol. 190:1094. PubMed Rouaud P, et al. 2014. J Exp Med. 211:975. 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: Shenoy AT, et al. 2022. Cytometry A. 101:892. PubMed Voss LF, et al. 2023. Front Immunol. 13:1021370. PubMed Rouaud P, et al. 2014. J Exp Med. 211:975. PubMed Kim CJ, et al. 2018. Immunity. 49:1034. PubMed Sarapulov AV, et al. 2020. Front Immunol. 11:599. PubMed Ly A, et al. 2020. Cell Reports. 29(8):2257-2269.e6.. PubMed Glaros V, et al. 2021. Immunity. 54:2005. PubMed Béguelin W, et al. 2020. Cancer Cell. 37(5):655-673. PubMed Haase P, et al. 2020. Eur J Immunol. 50:558. PubMed Tuong ZK, et al. 2021. Cell Rep. 37:110132. PubMed Katsuyama T, et al. 2019. J Clin Invest. 129:5411. PubMed
RRID: AB_10730618 (BioLegend Cat. No. 405717) AB_10730619 (BioLegend Cat. No. 405718)
Structure: Ig family
Distribution: B cells
Function: B cell differentiation
Cell Type: B cells
Biology Area: Immunology
Gene ID: 380797
UniProt: View information about IgD on UniProt.org
Clone: 11-26c.2a
Regulatory Status: RUO
Other Names: Immunoglobulin D
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.