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Alexa Fluor® 647 anti-mouse/human CD45R/B220 Antibody

Pricing & Availability
Clone
RA3-6B2 (See other available formats)
Regulatory Status
RUO
Other Names
B220
Isotype
Rat IgG2a, κ
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Product Citations
publications
1_RA3-6B2_AF647_090507
C57BL/6 mouse splenocytes stained with RA3-6B2 Alexa Fluor® 647
  • 1_RA3-6B2_AF647_090507
    C57BL/6 mouse splenocytes stained with RA3-6B2 Alexa Fluor® 647
  • 2_RA3-6B2_A647_B220_Antibody_IHC-F_080519
    C57BL/6 mouse frozen lymph node sections were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature and blocked with 5% FBS plus 5% rat serum for 1 hour at room temperature. Then the section was stained with 5 µg/mL of CD4 (clone GK1.5) Alexa Fluor® 594 (red), 5 µg/mL of B220 (clone RA3-6B2) Alexa Fluor® 647 (green) overnight at 4°C. The image was captured by 10X objective.
  • 3_Still-image_0316-5_20X-1_1
    Formalin-fixed, 300 micron-thick mouse spleen section was blocked, permeabilized and stained overnight with CD3 (clone 17A2) Alexa Fluor® 488 (red), CD21/35 (CR2/CR1)(clone 7E9) Alexa Fluor® 594 (green), and CD45R/B220 (clone RA3-6B2) Alexa Fluor® 647 (blue) all at 5 µg/mL, optically cleared, then analyzed at 215 μm imaging depth on a confocal microscope.
See Alexa Fluor® 647 spectral data
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103229 25 µg 81€
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103226 100 µg 186€
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Description

CD45R, also known as B220, is an isoform of CD45. It is a member of the protein tyrosine phosphatase (PTP) family with a molecular weight of approximately 180-240 kD. CD45R is expressed on B cells (at all developmental stages from pro-B cells through mature B cells), activated B cells, and subsets of T and NK cells. CD45R (B220) is also expressed on a subset of abnormal T cells involved in the pathogenesis of systemic autoimmunity in MRL-Faslpr and MRL-Fasgld mice. It plays a critical role in TCR and BCR signaling. The primary ligands for CD45 are galectin-1, CD2, CD3, and CD4. CD45R is commonly used as a pan-B cell marker; however, CD19 may be more appropriate for B cell specificity.

Product Details
Technical Data Sheet (pdf)

Product Details

Reactivity
Mouse,Human
Antibody Type
Monoclonal
Host Species
Rat
Immunogen
Abelson murine leukemia virus-induced pre-B tumor cells
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
IHC-F, 3D IHC - Verified
SB - Community verified
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 million cells in 100 µL volume. For immunohistochemistry on frozen tissue sections, a concentration range of 2.5 - 5.0 µg/mL is suggested. For immunofluorescence microscopy, a concentration range of 1.25 - 10 µg/mL is recommended. 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 633nm / 635nm.


Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.

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Excitation Laser
Red Laser (633 nm)
Application Notes

Clone RA3-6B2 has been described to react with an epitope on the extracellular domain of the transmembrane CD45 glycoprotein which is dependent upon the expression of exon A and specific carbohydrate residues. Additional reported applications (for the relevant formats) include: immunoprecipitation1, in vitro and in vivo modulation of B cell responses2-4, immunohistochemistry of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections5,6, and spatial biology (IBEX)14,15.

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
  1. Coffman RL. 1982. Immunol. Rev. 69:5. (IP)
  2. George A, et al. 1994. J. Immunol. 152:1014. (Activ)
  3. Asensi V, et al. 1989. Immunology 68:204. (Activ)
  4. Domiati-Saad R, et al. 1993. J. Immunol. 151:5936. (Activ)
  5. Hata H, et al. 2004. J. Clin. Invest. 114:582. (IHC)
  6. Monteith CE, et al. 1996. Can. J. Vet. Res. 60:193. (IHC)
  7. Shih FF, et al. 2006. J. Immunol. 176:3438. (FC)
  8. Chang C L-T, et al. 2007. J. Immunol. 178:6984.
  9. Fazilleau N, et al. 2007. Nature Immunol. 8:753.
  10. Lang GL, et al. 2008. Blood 111:2158. PubMed
  11. Charles N, et al. 2010. Nat. Med. 16:701. (FC) PubMed
  12. del Rio ML, et al. 2011. Transpl. Int. 24:501. (FC) PubMed
  13. Murakami R, et al. 2013. PLoS One. 8:73270. PubMed
  14. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
  15. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations
  1. Emgård J, et al. 2018. Immunity. 143:419. PubMed
  2. Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed
  3. Beura LK, et al. 2018. Immunity. 48:327. PubMed
  4. Hermetet F, et al. 2019. Nat Commun. 10:523. PubMed
  5. Ramakrishnan SK, et al. 2019. Nat Commun. 10:660. PubMed
  6. Cheng HW, et al. 2019. Nat Commun. 10:1739. PubMed
  7. Barry KC, et al. 2018. Nat Med. 24:1178. PubMed
  8. Farsakoglu Y et al. 2019. Cell reports. 26(9):2307-2315 . PubMed
  9. Simula L et al. 2018. Cell reports. 25(11):3059-3073 . PubMed
  10. Liu D et al. 2019. Immunity. 51(1):64-76 . PubMed
  11. Magri G et al. 2017. Immunity. 47(1):118-134 . PubMed
  12. Tsai F, et al. 2017. J Exp Med. 214:3753. PubMed
  13. Fallet B, et al. 2020. Cell Rep. 30:1013. PubMed
  14. LaFleur MW, et al. 2019. Nat Immunol. 20:1335. PubMed
  15. Sarapulov AV, et al. 2020. Front Immunol. 11:599. PubMed
  16. Chou YJ, et al. 2020. Sci Rep. 10:8422. PubMed
  17. Chauveau A, et al. 2020. Immunity. 52:794. PubMed
  18. Cao A, et al. 2018. Nat Commun. 9:3288. PubMed
  19. Yang J, et al. 2019. FASEB J. 33:12780. PubMed
  20. Lian J, et al. 2020. Cell Reports. 31(8):107679. PubMed
  21. Brownlie R, et al. 2008. J Exp Med. 205:883. PubMed
  22. Gil-Cruz C, et al. 2012. Proc Natl Acad Sci U S A. 109:1233. PubMed
  23. Harumiya S, et al. 2013. Arch Biochem Biophys. 533:18. PubMed
  24. Folgosa L, et al. 2013. J Immunol. 191:5951. PubMed
  25. Medgyesi D, et al. 2014. J Exp Med. 211:427. PubMed
  26. Neubert K, et al. 2014. J Immunol. 192:5830. PubMed
  27. Hoechst B, et al. 2016. J Immunol. 195:1517-1523. PubMed
  28. Kang S, et al. 2016. J Immunol. 196: 196 - 206. PubMed
  29. Yoshikawa S, et al. 2016. Sci Rep. 6:18738. PubMed
  30. Campisi L, et al. 2016. Nat Immunol. 10.1038/ni.3512. PubMed
  31. Prados A, Kollias G, Koliaraki V 2016. Sci Rep. 6: 33027. PubMed
  32. Hu HJ, et al. 2020. Cell Death Dis. 1.168055556. PubMed
  33. Cabañero D, et al. 2020. Elife. 9:00. PubMed
  34. Kim EH, et al. 2020. Elife. 9:00. PubMed
  35. Dammeijer F, et al. 2020. Cancer Cell. 38(5):685-700.e8. PubMed
  36. Webster HC, et al. 2020. J Immunol Methods. 112702:477. PubMed
  37. Dobosz E, et al. 2021. Dis Model Mech. :14. PubMed
  38. Vogel AB, et al. 2021. Nature. 592:283. PubMed
  39. Delvecchio FR, et al. 2021. Cell Mol Gastroenterol Hepatol. 12:1543. PubMed
  40. Jong RM, et al. 2022. J Immunol. 208:407. PubMed
  41. Wang Y, et al. 2021. Cancer Res. 81:174. PubMed
  42. Smith LK, et al. 2021. Elife. 10:. PubMed
  43. Chen C, et al. 2021. J Virol. 95:e0082921. PubMed
  44. Bangs DJ, et al. 2022. Cell Rep. 38:110266. PubMed
  45. Daniel CJ, et al. 2022. Mol Cancer Res. 20:1151. PubMed
  46. Cheng HW, et al. 2022. Nat Commun. 13:2027. PubMed
  47. Fleig S, et al. 2022. Nat Commun. 13:2022. PubMed
  48. Dai B, et al. 2022. Theranostics. 12:7603. PubMed
  49. Mi Z, et al. 2021. Vaccines (Basel). 10: . PubMed
  50. Dölz M, et al. 2022. iScience. 25:105372. PubMed
  51. Scherer S, et al. 2023. Nat Immunol. 24:501. PubMed
  52. Pineau J, et al. 2022. Elife. 11:. PubMed
  53. Schönberger K, et al. 2023. Anal Chem. 95:4325. PubMed
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RRID
AB_492875 (BioLegend Cat. No. 103229)
AB_389330 (BioLegend Cat. No. 103226)

Antigen Details

Structure
Protein tyrosine phosphatase (PTP) family, 180-240 kD
Distribution

B cells, T cell subset, NK cell subset

Function
Phosphatase, T and B cell activation
Ligand/Receptor
Galectin-1, CD2, CD3, CD4
Cell Type
B cells, NK cells, T cells
Biology Area
Cell Biology, Immunology, Inhibitory Molecules, Neuroscience, Neuroscience Cell Markers
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 View all products for this Gene ID 5788 View all products for this Gene ID
UniProt
View information about CD45R on UniProt.org

Related FAQs

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

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.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

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.

Are other fluorophores compatible with IBEX?

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.

The same antibody works in one tissue type but not another. What is happening?

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.

How can I be sure the staining I’m seeing in my tissue is real?

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.

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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