Biolegend, 127636, Alexa Fluor® 594 anti-mouse Ly-6G Antibody, 100μg

Lymphocyte antigen 6 complex, locus G (Ly-6G), a 21-25 kD GPI-anchored protein, is expressed on the majority of myeloid cells in bone marrow and peripheral granulocytes.
100μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: Ly-6G transfected EL-4J cell line.
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 SB - Community verified
Recommended Usage: Each lot of this antibody is quality control tested by immunohistochemical staining on frozen tissue sections. For immunohistochemistry, a concentration range of 2.0 - 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
Excitation Laser: Green Laser (532 nm)/Yellow-Green Laser (561 nm)
Application Notes: While 1A8 recognizes only Ly-6G, clone RB6-8C5 recognizes both Ly-6G and Ly-6C. Clone RB6-8C5 binds with high affinity to mouse Ly-6G molecules and to a lower extent to Ly-6C15. Clone RB6-8C5 impairs the binding of anti-mouse Ly-6G clone 1A815. However, clone RB6-8C5 is able to stain in the presence of anti-mouse Ly-6C clone HK1.416. Additional reported applications (for the relevant formats) include: immunohistochemistry9 of frozen sections10 and paraffin-embedded sections11, depletion4, 12-14, and spatial biology (IBEX)20,21. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for in vivo studies or highly sensitive assays (Cat. No. 127632, 127649, 127650, 127661 and 127662).
Additional Product Notes: This product has been verified for IHC-F (Immunohistochemistry - frozen tissue sections) and IHC-P (Immunohistochemistry - formalin-fixed paraffin-embedded tissues) on the NanoString GeoMx® Digital Spatial Profiler. The GeoMx® enables researchers to perform spatial analysis of protein and RNA targets in FFPE and fresh frozen human and mouse samples. For more information about our spatial biology products and the GeoMx® platform, please visit our spatial biology page. View more applications data for this product in our Scientific Poster Library.
Application References(PubMed link indicates BioLegend citation): Fleming TJ, et al. 1993. J. Immunol. 151:2399. (FC) Daley JM, et al. 2008. J. Leukocyte Biol. 83:1. (FC) Dietlin TA, et al. 2007. J. Leukocyte Biol. 81:1205. (FC) Daley J, et al. 2007. J. Leukocyte Biol. doi:10.1189. (Deplete) PubMed Tadagavadi RK, et al. 2010. J. Immunol. 185:4904. PubMed Sumagin R, et al. 2010. J. Immunol. 185:7057. PubMed Guiducci C, et al. 2010. J. Exp Med. 207:2931. PubMed Fujita M, et al. 2011. Cancer Res. 71:2664. PubMed Van Leeuwen, et al. 2008. Arterioscler. Thromb. Vasc. Biol. 28:84. (IHC) Kowanetz M, et al. 2010. P. Natl. Acad. Sci. USA 107:21248. [supplementary data] (IHC) Esbona K, et al. 2016. Breast Cancer Res. 18:35. (IHC) Wojtasiak M, et al. 2010. J. Gen. Virol. 91:2158. (FC, Deplete) Jaeger BN, et al. 2012. J. Exp. Med. 209:565. (Deplete) Wozniak KL, et al. 2012. BMC Immunol. 13:65 (FC, Deplete) Ribechini E, et al. 2009. Eur. J. Immunol. 39:3538. Ng LG, et al. 2011. J Invest. Dermatol. 131:2058. PubMed Ma C, et al. 2012. J. Leukoc. Biol. 92:1199. McCartney-Francis, N, et al. 2014. J Leukoc. Biol. 96:917. PubMed Her Z, et al. 2014. EMBO Mol. Med. 7:24. 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: Bellut M, et al. 2023. J Neuroinflammation. 20:4. PubMed Pernet E, et al. 2023. Nature. 614:530. PubMed Choudhury SR, et al. 2020. Cell. 178(5):1205-1221.e17.. PubMed Qi JL, et al. 2020. MedComm (Beijing). 1:188. PubMed Ueki H, et al. 2020. Nat Protoc. 15:1041. PubMed Ouyang J, et al. 2020. Biomed Res Int. 4307385:2020. PubMed Pitsch J, et al. 2021. Ann Neurol. 89:666. PubMed Moniaga C, et al. 2015. Sci Rep. 5: 15319. PubMed Simões FC, et al. 2020. Nat Commun. 0.875. PubMed Li MY, et al. 2021. Developmental Cell. 56:2547. PubMed Chmielewski M and Abken H 2017. Cell Rep.. 10.1016/j.celrep.2017.11.063. PubMed Lee EKS, et al. 2018. Cell Host Microbe. 23:121. PubMed Wang F, et al. 2021. Oxid Med Cell Longev. 2021:5833857. PubMed
RRID: AB_2563207 (BioLegend Cat. No. 127636)
Structure: A 21-35 kD GPI-anchorded membrane protein
Distribution: Expressed on the majority of myeloid cells in bone marrow and peripheral granulocytes. The monoclonal antibody RB6-8C5 recognizes both Ly-6G and Ly-6C.
Cell Type: Granulocytes, Macrophages, Monocytes
Biology Area: Immunology, Innate Immunity
Antigen References: Fleming TJ, et al. 1993. J. Immunol. 151:2399.
Gene ID: 546644
UniProt: View information about Ly-6G on UniProt.org
Clone: 1A8
Regulatory Status: RUO
Other Names: Lymphocyte antigen 6 complex, locus G
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.