Biolegend, 103124, Alexa Fluor® 647 anti-mouse CD45 Antibody, 100μg

CD45 is a 180-240 kD glycoprotein also known as the leukocyte common antigen (LCA), T200, or Ly-5. It is a member of the protein tyrosine phosphatase (PTP) family, expressed on all hematopoietic cells except mature erythrocytes and platelets. There are different isoforms of CD45 that arise from variable splicing of exons 4, 5, and 6, which encode A, B, and C determinants, respectively. CD45 plays a key role in TCR and BCR signal transduction. These isoforms are very specific to the activation and maturation state of the cell as well as cell type. The primary ligands for CD45 are galectin-1, CD2, CD3, CD4, TCR, CD22, and Thy-1.
100μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: Mouse thymus or spleen
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 ICC, 3D IHC - Verified SB - Community verified IHC, 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 106 cells in 100 µl volume. For 3D immunohistochemistry on formalin-fixed tissues, a concentration of 5.0 µg/mL is suggested. It is recommended that the reagent be titrated for optimal performance for other applications. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633nm / 635nm. 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: Clone 30-F11 reacts with all isoforms and both CD45.1 and CD45.2 alloantigens of CD45. Additional reported applications (for relevant formats) include: immunoprecipitation3, complement-dependent cytotoxicity1,5, immunohistochemistry (acetone-fixed frozen sections, zinc-fixed paraffin-embedded sections and formalin-fixed paraffin-embedded sections)4,6, Western blotting7, and spatial biology (IBEX)10,11. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. No. 103163 and 103164).
Additional Product Notes: For use in spatial biology, this antibody has been demonstrated for use in immunohistochemistry using IBEX (Reported in the literature, not verified in house) and the NanoString GeoMx® Digital Spatial Profiler. IBEX: 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). NanoString GeoMx®: This product has been verified for IHC-F (Immunohistochemistry - frozen tissue sections) 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.
Application References(PubMed link indicates BioLegend citation): Podd BS, et al. 2006. J. Immunol. 176:6532. (FC, CMCD) PubMed Haynes NM, et al. 2007. J. Immunol. 179:5099. (FC) Ledbetter JA, et al. 1979. Immunol. Rev. 47:63. (IP) Simon DI, et al. 2000. J. Clin. Invest. 105:293. (IHC) Seaman WE. 1983. J. Immunol. 130:1713. (CMCD) Cornet A, et al. 2001. P. Natl. Acad. Sci. USA 98:13306. (IHC) Tsuboi S and Fukuda M. 1998. J. Biol. Chem. 273:30680. (WB) PubMed Liu F, et al. 2012. Blood. 119:3295. PubMed Pelletier AN, et al. 2012. J. Immunol. 188:5561. 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: Osterloh A, et al. 2016. Infect Immun . . PubMed Moderzynski K, et al. 2016. PLoS Negl Trop Dis. . PubMed Chen W, et al. 2023. JCI Insight. 8:. PubMed Iguchi A, et al. 2023. iScience. 26:106375. PubMed Yan Y, et al. 2021. Immunity. 54:499. PubMed Yamamoto A, et al. 2022. Arthritis Rheumatol. 74:860. PubMed Frederico B, et al. 2022. Dev Cell. 57:1957. PubMed Cho J, et al. 2016. Nat Commun. 7:13373. PubMed Donato C, et al. 2020. Cell Reports. 32(10):108105. PubMed Boal-Carvalho I, et al. 2020. EMBO Rep. 21:e50421. PubMed Hu X, et al. 2016. Nat Commun. 7:13095. PubMed Moore SM, et al. 2019. Transl Psychiatry. 9:24. PubMed Biffi G, et al. 2018. Cancer Discov. 2:282. PubMed Hu K, et al. 2015. PLoS One. 10: 0137123. PubMed Perner C, et al. 2020. Immunity. 53(5):1063-1077.e7. PubMed Sutton NR, et al. 2019. Arterioscler Thromb Vasc Biol. 40:61. PubMed Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed Fang EF, et al. 2019. Nat Neurosci. 22:401. PubMed Ruhland MK, et al. 2020. Cancer Cell. 37(6):786-799.e5. PubMed Piedrafita G, et al. 2020. Nat Commun. 11:1429. PubMed Uceda-Castro R, et al. 2022. Cell Rep Med. 3:100821. PubMed Donato C, et al. 2021. STAR Protocols. 2(2):100480. PubMed Pineda CM, et al. 2019. J Cell Biol. 218:3212. PubMed Kang MH, et al. 2020. Nat Commun. 3.186805556. PubMed Kim S, et al. 2022. Redox Biol. 54:102347. PubMed Levesque S, et al. 2019. Oncoimmunology. 8:e1657375. PubMed Kuhn JA, et al. 2021. Elife. 10:. PubMed Simões FC, et al. 2020. Nat Commun. 0.875. PubMed Tummers B, et al. 2020. Immunity. 52(6):994-1006.e8. PubMed Huang L, et al. 2018. Exp Neurol. 300:41. PubMed Sakamoto K, et al. 2021. Immunity. 54:2321. PubMed Wang S, et al. 2021. Commun Biol. 22:. PubMed Shibuya M, et al. 2021. iScience. 24:103131. PubMed de Winde CM, et al. 2021. J Cell Sci. 134: . PubMed Ovadya Y, et al. 2018. Nat Commun. 9:5435. PubMed Yomtoubian S, et al. 2020. Cell Reports. 30(3):755-770.e6.. PubMed Lu YJ, et al. 2021. Cell Rep. 36:109696. PubMed Thacker VV, et al. 2020. Elife. 9:00. PubMed Sanders K, et al. 2015. Cancer Immunol Res. 3: 891-901. PubMed Clemente–Casares X, et al. 2017. Immunity. 47:974. PubMed Bakalar MH et al. 2018. Cell. 174(1):131-142 . PubMed Chen W, et al. 2016. Nat Commun. 7: 11302. PubMed Xiong J et al. 2018. Molecular cell. 69(4):689-698 . PubMed Williams IM, et al. 2018. J Clin Invest. 128:699. PubMed Koyama M et al. 2019. Immunity. 51(5):885-898 . PubMed Oni TE, et al. 2020. J Exp Med. :217. PubMed Prabakaran T, et al. 2021. EBioMedicine. 66:103314. PubMed
RRID: AB_493534 (BioLegend Cat. No. 103123) AB_493533 (BioLegend Cat. No. 103124)
Structure: Protein tyrosine phosphatase (PTP) family, 180-240 kD
Distribution: All hematopoietic cells except mature erythrocytes and platelets
Function: Phosphatase, T and B cell activation
Ligand/Receptor: Galectin-1, CD2, CD3, CD4, TCR, CD22, Thy-1
Cell Type: B cells, Dendritic cells, Mesenchymal Stem Cells, Tregs
Biology Area: Cell Biology, Immunology, Inhibitory Molecules, Innate Immunity, Neuroscience, Neuroscience Cell Markers, Stem Cells
Molecular Family: CD Molecules
Antigen References: 1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press. 2. Trowbridge IS, et al. 1993. Annu. Rev. Immunol. 12:85. 3. Kishihara K, et al. 1993. Cell 74:143. 4. Pulido R, et al. 1988. J. Immunol. 140:3851.
Gene ID: 19264
UniProt: View information about CD45 on UniProt.org
Clone: 30-F11
Regulatory Status: RUO
Other Names: T200, Ly-5, LCA
Isotype: Rat IgG2b, 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.