Alexa Fluor® 647 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.

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 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

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)

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. Pineau J, et al. 2022. Elife. 11:. PubMed
2. Schönberger K, et al. 2023. Anal Chem. 95:4325. PubMed
3. Seal R, et al. 2023. Front Immunol. 14:1179311. PubMed
4. Wang Y, et al. 2021. Cancer Res. 81:174. PubMed
5. Mi Z, et al. 2021. Vaccines (Basel). 10: . PubMed
6. Dölz M, et al. 2022. iScience. 25:105372. PubMed
7. Scherer S, et al. 2023. Nat Immunol. 24:501. PubMed
8. Cabañero D, et al. 2020. Elife. 9:00. PubMed
9. Jong RM, et al. 2022. J Immunol. 208:407. PubMed
10. Delvecchio FR, et al. 2021. Cell Mol Gastroenterol Hepatol. 12:1543. PubMed
11. Kim EH, et al. 2020. Elife. 9:00. PubMed
12. Kang S, et al. 2016. J Immunol. 196: 196 - 206. PubMed
13. Hu HJ, et al. 2020. Cell Death Dis. 1.168055556. PubMed
14. Barry KC, et al. 2018. Nat Med. 24:1178. PubMed
15. Farsakoglu Y et al. 2019. Cell reports. 26(9):2307-2315 . PubMed
16. Chou YJ, et al. 2020. Sci Rep. 10:8422. PubMed
17. Brownlie R, et al. 2008. J Exp Med. 205:883. PubMed
18. Bangs DJ, et al. 2022. Cell Rep. 38:110266. PubMed
19. Neubert K, et al. 2014. J Immunol. 192:5830. PubMed
20. Webster HC, et al. 2020. J Immunol Methods. 112702:477. PubMed
21. LaFleur MW, et al. 2019. Nat Immunol. 20:1335. PubMed
22. Dai B, et al. 2022. Theranostics. 12:7603. PubMed
23. Folgosa L, et al. 2013. J Immunol. 191:5951. PubMed
24. Sarapulov AV, et al. 2020. Front Immunol. 11:599. PubMed
25. Medgyesi D, et al. 2014. J Exp Med. 211:427. PubMed
26. Emgård J, et al. 2018. Immunity. 143:419. PubMed
27. Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed
28. Daniel CJ, et al. 2022. Mol Cancer Res. 20:1151. PubMed
29. Fallet B, et al. 2020. Cell Rep. 30:1013. PubMed
30. Liu D et al. 2019. Immunity. 51(1):64-76 . PubMed
31. Cheng HW, et al. 2019. Nat Commun. 10:1739. PubMed
32. Yang J, et al. 2019. FASEB J. 33:12780. PubMed
33. Lian J, et al. 2020. Cell Reports. 31(8):107679. PubMed
34. Vogel AB, et al. 2021. Nature. 592:283. PubMed
35. Dobosz E, et al. 2021. Dis Model Mech. :14. PubMed
36. Chauveau A, et al. 2020. Immunity. 52:794. PubMed
37. Magri G et al. 2017. Immunity. 47(1):118-134 . PubMed
38. Tsai F, et al. 2017. J Exp Med. 214:3753. PubMed
39. Simula L et al. 2018. Cell reports. 25(11):3059-3073 . PubMed
40. Hermetet F, et al. 2019. Nat Commun. 10:523. PubMed
41. Chen C, et al. 2021. J Virol. 95:e0082921. PubMed
42. Beura LK, et al. 2018. Immunity. 48:327. PubMed
43. Ramakrishnan SK, et al. 2019. Nat Commun. 10:660. PubMed
44. Cheng HW, et al. 2022. Nat Commun. 13:2027. PubMed
45. Fleig S, et al. 2022. Nat Commun. 13:2022. PubMed
46. Gil-Cruz C, et al. 2012. Proc Natl Acad Sci U S A. 109:1233. PubMed
47. Hoechst B, et al. 2016. J Immunol. 195:1517-1523. PubMed
48. Cao A, et al. 2018. Nat Commun. 9:3288. PubMed
49. Smith LK, et al. 2021. Elife. 10:. PubMed
50. Prados A, Kollias G, Koliaraki V 2016. Sci Rep. 6: 33027. PubMed
51. Harumiya S, et al. 2013. Arch Biochem Biophys. 533:18. PubMed
52. Dammeijer F, et al. 2020. Cancer Cell. 38(5):685-700.e8. PubMed
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54. Yoshikawa S, et al. 2016. Sci Rep. 6:18738. PubMed

RRID

AB_389330 (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

Specificity (DOES NOT SHOW ON TDS):

CD45R

Specificity Alt (DOES NOT SHOW ON TDS):

CD45R/B220

App Abbreviation (DOES NOT SHOW ON TDS):

FC,IHC-F,3D IHC,SB

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
• Immunocytochemistry 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