Brilliant Violet 510™ anti-mouse/human CD45R/B220 Antibody

Product Details

Verified Reactivity

Mouse, Human

Reported Reactivity

Cat

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 and BSA (origin USA).

Preparation

The antibody was purified by affinity chromatography and conjugated with Brilliant Violet 510™ under optimal conditions.

Concentration

µg sizes: 0.2 mg/mL
µL sizes: lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

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, IHC-F - 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 using the µl size, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. For flow cytometric staining using the µg size, 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. It is recommended that the reagent be titrated for optimal performance for each application.

Brilliant Violet 510™ excites at 405 nm and emits at 510 nm. The bandpass filter 510/50 nm is recommended for detection, although filter optimization may be required depending on other fluorophores used. Be sure to verify that your cytometer configuration and software setup are appropriate for detecting this channel. Refer to your instrument manual or manufacturer for support. Brilliant Violet 510™ is a trademark of Sirigen Group Ltd.

Learn more about Brilliant Violet™.

This product is subject to proprietary rights of Sirigen Inc. and is made and sold under license from Sirigen Inc. The purchase of this product conveys to the buyer a non-transferable right to use the purchased product for research purposes only. This product may not be resold or incorporated in any manner into another product for resale. Any use for therapeutics or diagnostics is strictly prohibited. This product is covered by U.S. Patent(s), pending patent applications and foreign equivalents.

Excitation Laser

Violet Laser (405 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 responses^2-4, immunohistochemistry of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections^5,6, and spatial biology (IBEX)^14,15.

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)

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. Hutter K, et al. 2022. Front Immunol. 13:967914. PubMed
2. Chaurio RA, et al. 2022. Immunity. 55:115. PubMed
3. Alam A, et al. 2022. Cancer Cell. 40:153. PubMed
4. Molina MS, et al. 2022. PLoS One. 17:e0273075. PubMed
5. Noir S, et al. 2017. Nucleic Acids Res. 10.1093/nar/gkx203. PubMed
6. Tan J, et al. 2021. iScience. 24(8):102835. PubMed
7. Vono M, et al. 2020. Cell Reports. 28(7):1773-1784.e5.. PubMed
8. Christian DA, et al. 2022. Sci Immunol. 7:eabq7432. PubMed
9. Read KA, et al. 2023. Nat Commun. 14:1652. PubMed
10. Zhang YN, et al. 2023. Nat Commun. 14:1985. PubMed
11. Yazicioglu YF, et al. 2023. Nat Immunol. 24:991. PubMed
12. Gao Y, et al. 2023. iScience. 26:106729. PubMed
13. Shibata K, et al. 2022. Nat Commun. 13:6948. PubMed
14. Lentucci C, et al. 2017. J Biol Chem. 292:2754-2772. PubMed
15. Zhang J, et al. 2021. MedComm (Beijing). 2:256. PubMed
16. Webb LMC, et al. 2021. Aging Cell. 20:e13295. PubMed
17. Barry KC, et al. 2018. Nat Med. 24:1178. PubMed
18. Shi B, et al. 2018. J Immunol. 200:586. PubMed
19. Franks SE, et al. 2019. J Immunol. 202:3381. PubMed
20. Xu AQ, et al. 2020. Elife. 9:00. PubMed
21. Pan H, et al. 2020. Mol Psychiatry. . PubMed
22. Yen WF et al. 2019. Cell reports. 27(5):1472-1486 . PubMed
23. Huang L, et al. 2017. PLoS Biol.. 10.1371/journal.pbio.2001750. PubMed
24. Yang L, et al. 2021. Cell Death Differ. 28:2616. PubMed
25. Sato R, et al. 2020. Int Immunol. 499:32. PubMed
26. Zbesko JC, et al. 2021. Brain Behav Immun. 91:578. PubMed
27. Zhu Y, et al. 2022. Clin Transl Med. 12:e887. PubMed
28. Hutter K, et al. 2020. FEBS J. . PubMed
29. Webster P, et al. 2018. Nat Commun. 9:2649. PubMed
30. Dietmar Herndler‐Brandstetter et al. 2018. Immunity. 48(4):716-729 . PubMed
31. Mulas F, et al. 2020. Cell Mol Immunol. . PubMed
32. Kurniawan H, et al. 2020. Cell Metabolism. 31(5):920-936. PubMed
33. Schoeler K, et al. 2019. FEBS J. 10.1111/febs.14934. PubMed
34. Reismann D, et al. 2017. Nat Commun.. 10.1038/s41467-017-01538-9. PubMed
35. Chauveau L, et al. 2021. EMBO Rep. 22:e52447. PubMed
36. Grioni M, et al. 2021. Blood Adv. 5:2817. PubMed
37. Matundan H, et al. 2019. J Virol. 93. PubMed
38. Walsh SM, et al. 2021. eLife. 10:00. PubMed
39. Matsumura T, et al. 2022. Nat Commun. 13:7064. PubMed
40. Lehrke MJ, et al. 2021. Elife. 10:. PubMed
41. Zhang YN, et al. 2021. Sci Adv. 7:eabj3107. PubMed
42. Schager AE, et al. 2018. MBio. 9:02379. PubMed
43. Barbet G, et al. 2018. Immunity. 48:584. PubMed
44. Molina MS, et al. 2021. Front Immunol. 12:699128. PubMed
45. Yamada H, et al. 2018. J Immunol. 201:3492. PubMed
46. Werner A, et al. 2021. iScience. 24:103076. PubMed
47. Tähtinen S, et al. 2022. Nat Immunol. 23:532. PubMed
48. Zhang Y, et al. 2022. Front Pharmacol. 13:952775. PubMed
49. Rodrigues KA, et al. 2021. Sci Adv. 7:eabj6538. PubMed
50. Orr MT, et al. 2019. NPJ Vaccines. 4:1. PubMed
51. Jing Y, et al. 2019. J Allergy Clin Immunol. 144:1377. PubMed
52. Bastow CR, et al. 2022. Front Immunol. 13:873586. PubMed
53. Du Z, et al. 2020. J Allergy Clin Immunol. . PubMed
54. Lucas ED, et al. 2020. Cell Reports. 33(2):108258. PubMed
55. Campisi L, et al. 2016. Nat Immunol. 10.1038/ni.3512. PubMed
56. Abdel Malik R, et al. 2017. Circ Res. 120:99. PubMed

RRID

AB_2561394 (BioLegend Cat. No. 103247)
AB_2561394 (BioLegend Cat. No. 103248)

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

Documentation

• Technical data sheet
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Immunohistochemistry Protocol for Frozen Sections
• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

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.

Other Formats

Certificate of Analysis