Biolegend, 139322, Alexa Fluor® 647 anti-mouse CD64 (FcγRI) Antibody, 100μg

CD64 is a 72 kD single chain type I glycoprotein also known as FcγRI and FcRI. CD64 is a member of the immunoglobulin superfamily and is expressed on monocytes/macrophages, dendritic cells, and mast cells. The expression can be upregulated by IFN-γ stimulation. CD64 binds IgG immune complex. It plays a role in antigen capture, phagocytosis of IgG/antigen complexes, and antibody-dependent cellular cytotoxicity (ADCC).
100μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: BALB/c mouse FcγRI-human IgG Fc fusion protein.
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 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® 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 X54-5/7.1 antibody reacts with mouse strains carrying CD64a and b alleles but not CD64d. X54-5/7.1 recognizes a conformational determinant formed between domains 2 and 3. Additional reported application (for relevant formats) include: immunoprecipitation1, and spatial biology (IBEX)5,6. Clone X54-5/7.1 is not found to be useful for Western blots1.
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): Tan PS, et al. 2003. J. Immunol. 170:2549. (IP) Ingersoll MA, et al. 2010. Blood 115:e10. (FC) Ozeri E, et al. 2012. J. Immunol. 189:146. PubMed Richardson ML, et al. 2014. PLoS Negl Trop Dis. 8:2825. 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: Peterson EJ, et al. 2019. Mol Syst Biol. 15:e8584. PubMed Sierro F, et al. 2017. Immunity. 47:374. PubMed Ahmed A, et al. 2023. Mol Oncol. 1545:17. PubMed Sepe JJ, et al. 2022. JACC Basic Transl Sci. 7:915. PubMed Wieghofer P, et al. 2021. EMBO J. 40:e105123. PubMed Ferrer-Font L, et al. 2020. Elife. 9:. PubMed
RRID: AB_2566560 (BioLegend Cat. No. 139321) AB_2566561 (BioLegend Cat. No. 139322)
Structure: Ig superfamily, type I glycoprotein, 72 kD
Distribution: Monocytes, macrophages, mast cells, dendritic cells
Function: Phagocytosis, ADCC
Ligand/Receptor: IgG
Cell Type: Dendritic cells, Macrophages, Mast cells, Monocytes
Biology Area: Immunology, Innate Immunity
Molecular Family: CD Molecules, Fc Receptors
Gene ID: 14129
UniProt: View information about CD64 on UniProt.org
Clone: X54-5/7.1
Regulatory Status: RUO
Other Names: FcRI
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.