C57BL/6 mouse frozen lymph node section was 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 2.5 µg/ml of B220 (clone RA3-6B2) Alexa Fluor® 594 (red), 2.5 µg/ml of CD4 (clone RM4-5) Alexa Fluor® 488 (green), and 2.5 µg/ml of CD8 (clone 53-6.7) Alexa Fluor® 647 (blue) overnight at 4°C. The image was captured by 10X objective.
C57BL/6 mouse frozen lymph node section was 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 2.5 µg/ml of B220 (clone RA3-6B2) Alexa Fluor® 594 (red), 2.5 µg/ml of CD4 (clone RM4-5) Alexa Fluor® 488 (green), and 2.5 µg/ml of CD8 (clone 53-6.7) Alexa Fluor® 647 (blue) overnight at 4°C. The image was captured by 10X objective.
C57BL/6 mouse splenocytes were stained with CD45R/B220 (clone RA3-6B2) Alexa Fluor® 594 (filled histogram) or rat IgG2a, κ Alexa Fluor® 594 isotype control (open histogram). The data was acquired by BD LSRFortessa™ cell analyzer equipped with Yellow-Green Laser (561 nm).
Formalin-fixed, 300 micron-thick mouse spleen section was blocked, permeabilized and stained overnight with CD4 (clone RM4-5) Alexa Fluor® 647 (red), CD169 (Siglec-1)(clone 3D6.112) Alexa Fluor® 488 (green), and CD45R/B220 (clone RA3-6B2) Alexa Fluor® 594 (blue) all at 5 µg/mL, optically cleared, then analyzed at 220 μm imaging depth on a confocal microscope. Watch the video.
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.
Each lot of this antibody is quality control tested by immunofluorescence staining. For immunohistochemistry, a concentration range of 2.5-5 μg/ml is recommended. For immunofluorescence microscopy, a concentration range of 1.25-10 μg/ml is recommended. For flow cytometric staining, the suggested use of this reagent is ≤0.25 µg per million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.
* Alexa Fluor® 594 has an excitation maximum of 590 nm, and a maximum emission of 617 nm.
Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.
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, and immunohistochemistry of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections5,6.
Application References
(PubMed link indicates BioLegend citation)
Coffman RL. 1982. Immunol. Rev. 69:5. (IP)
George A, et al. 1994. J. Immunol. 152:1014. (Activ)
Asensi V, et al. 1989. Immunology 68:204. (Activ)
Domiati-Saad R, et al. 1993. J. Immunol. 151:5936. (Activ)
Hata H, et al. 2004. J. Clin. Invest. 114:582. (IHC)
Monteith CE, et al. 1996. Can. J. Vet. Res. 60:193. (IHC)
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.
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