Alexa Fluor® 647 anti-mouse CD45 Antibody

Product Details

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 cytotoxicity^1,5, 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)

1. Podd BS, et al. 2006. J. Immunol. 176:6532. (FC, CMCD) PubMed
2. Haynes NM, et al. 2007. J. Immunol. 179:5099. (FC)
3. Ledbetter JA, et al. 1979. Immunol. Rev. 47:63. (IP)
4. Simon DI, et al. 2000. J. Clin. Invest. 105:293. (IHC)
5. Seaman WE. 1983. J. Immunol. 130:1713. (CMCD)
6. Cornet A, et al. 2001. P. Natl. Acad. Sci. USA 98:13306. (IHC)
7. Tsuboi S and Fukuda M. 1998. J. Biol. Chem. 273:30680. (WB) PubMed
8. Liu F, et al. 2012. Blood. 119:3295. PubMed
9. Pelletier AN, et al. 2012. J. Immunol. 188:5561. PubMed
10. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
11. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

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5. Yan Y, et al. 2021. Immunity. 54:499. PubMed
6. Yamamoto A, et al. 2022. Arthritis Rheumatol. 74:860. PubMed
7. Frederico B, et al. 2022. Dev Cell. 57:1957. PubMed
8. Donato C, et al. 2020. Cell Reports. 32(10):108105. PubMed
9. Cho J, et al. 2016. Nat Commun. 7:13373. PubMed
10. Boal-Carvalho I, et al. 2020. EMBO Rep. 21:e50421. PubMed
11. Moore SM, et al. 2019. Transl Psychiatry. 9:24. PubMed
12. Hu X, et al. 2016. Nat Commun. 7:13095. PubMed
13. Biffi G, et al. 2018. Cancer Discov. 2:282. PubMed
14. Hu K, et al. 2015. PLoS One. 10: 0137123. PubMed
15. Sutton NR, et al. 2019. Arterioscler Thromb Vasc Biol. 40:61. PubMed
16. Perner C, et al. 2020. Immunity. 53(5):1063-1077.e7. PubMed
17. Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed
18. Fang EF, et al. 2019. Nat Neurosci. 22:401. PubMed
19. Piedrafita G, et al. 2020. Nat Commun. 11:1429. PubMed
20. Ruhland MK, et al. 2020. Cancer Cell. 37(6):786-799.e5. PubMed
21. Uceda-Castro R, et al. 2022. Cell Rep Med. 3:100821. PubMed
22. Pineda CM, et al. 2019. J Cell Biol. 218:3212. PubMed
23. Donato C, et al. 2021. STAR Protocols. 2(2):100480. PubMed
24. Kang MH, et al. 2020. Nat Commun. 3.186805556. PubMed
25. Kim S, et al. 2022. Redox Biol. 54:102347. PubMed
26. Levesque S, et al. 2019. Oncoimmunology. 8:e1657375. PubMed
27. Kuhn JA, et al. 2021. Elife. 10:. PubMed
28. Simões FC, et al. 2020. Nat Commun. 0.875. PubMed
29. Huang L, et al. 2018. Exp Neurol. 300:41. PubMed
30. Tummers B, et al. 2020. Immunity. 52(6):994-1006.e8. PubMed
31. Sakamoto K, et al. 2021. Immunity. 54:2321. PubMed
32. Wang S, et al. 2021. Commun Biol. 22:. PubMed
33. Shibuya M, et al. 2021. iScience. 24:103131. PubMed
34. de Winde CM, et al. 2021. J Cell Sci. 134: . PubMed
35. Ovadya Y, et al. 2018. Nat Commun. 9:5435. PubMed
36. Yomtoubian S, et al. 2020. Cell Reports. 30(3):755-770.e6.. PubMed
37. Lu YJ, et al. 2021. Cell Rep. 36:109696. PubMed
38. Clemente–Casares X, et al. 2017. Immunity. 47:974. PubMed
39. Bakalar MH et al. 2018. Cell. 174(1):131-142 . PubMed
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41. Sanders K, et al. 2015. Cancer Immunol Res. 3: 891-901. PubMed
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RRID

AB_493533 (BioLegend Cat. No. 103123)
AB_493533 (BioLegend Cat. No. 103124)

Antigen Details

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 View all products for this Gene ID

Specificity (DOES NOT SHOW ON TDS):

CD45

Specificity Alt (DOES NOT SHOW ON TDS):

CD45

App Abbreviation (DOES NOT SHOW ON TDS):

FC,ICC,IHC,3D IHC,SB

UniProt

View information about CD45 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol
• Ce3D™ Tissue Clearing Kit

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