Spleens and lymph nodes were fixed for 2–4 h in 4% PFA in PBS, washed, and incubated overnight in 30% sucrose in PBS. Tissues were embedded in OCT and sectioned using a cryostat. Sections were blocked for 30 min in staining solution (5% donkey serum in PBS-Tween) containing FcRblock and subsequently incubated overnight at 4°C in staining solution with 1–2 ng/µl anti–CD3-AlexaFluor 647 (Clone 17A2, Biolegend; Cat. #100209), anti-B220-Brilliant Violet 421 (Clone RA3-6B2, Biolegend; Cat. #103239), and rabbit anti-laminin 1+2 (Abcam; Cat. #AB7463) labeled with CF568 Mix-n-stain kit (Biotium). After washing in PBS, slides were mounted using ProLong Diamond antifade (Invitrogen) and imaged at room temperature using a Zeiss LSM710 AxioObserver inverted confocal microscope. Images were captured using Zen 2012 (Zeiss) software and analyzed with Volocity v6.3 (Perkin Elmer) software with γ = 1.00.
Anti b220 brilliant violet 421
Manufactured by BioLegend
The Anti-B220 Brilliant Violet 421 is a fluorochrome-conjugated antibody that binds to the B220 antigen, also known as CD45R. It is commonly used in flow cytometry applications to identify and characterize B cells.
Automatically generated - may contain errors
Lab products found in correlation
Cm1850 cryostat, by Leica (2 mentions)
Anti f4 80 pe, by BioLegend (2 mentions)
Clone bm8, by BioLegend (2 mentions)
Anti cd45.1 alexa 647, by BioLegend (2 mentions)
Fv1200 laser scanning confocal microscope, by Olympus (2 mentions)
Clone ra3 6b2, by BioLegend (2 mentions)
Volocity v6, by PerkinElmer (1 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (1 mentions)
Lsm 710 axioobserver, by Zeiss (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Anti cd3 alexafluor 647, by BioLegend (1 mentions)
Rabbit anti laminin 1 2, by Abcam (1 mentions)
3 protocols using anti b220 brilliant violet 421
1
Immunofluorescent Tissue Staining and Imaging
2
Multicolor Immunofluorescence Staining of Spleen
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Spleens were fixed with 2.5% neutral buffered formalin (NBF) overnight at 4°C and then equilibrated in 30% sucrose solution for 24h at 4°C. Spleens were embedded with tissue freezing medium (Triangle Biomedical Sciences). Cryosections were cut using a Leica CM1850 cryostat at a thickness of 30 μm. Sections were washed and stained in PBS containing 5 μM EDTA and 2% FBS. Anti-CD45.1 Alexa-647 (0.4 μg/ml; clone A20; BioLegend), anti-B220 Brilliant Violet 421 (0.5 μg/ml clone RA3-6B2, BioLegend) and anti-F4/80 PE (0.5 μg/ml clone BM8, BioLegend) were used for staining. Primary antibodies were incubated on cryosections overnight at 4°C. After staining, sections were washed 4-5 times in staining buffer. 2-3 drops of FluorSave Reagent (Calbiochem) were added to each section before addition of a coverslip. Images were acquired using an Olympus FV1200 laser scanning confocal microscope equipped 4 detectors, 6 laser lines (405, 458, 488, 515, 559, and 635 nm) and 5 objectives (4x/0.16 NA, 10x/0.4 NA, 20x/0.75 NA, 40x/0.95 NA, and chromatic aberration corrected 60x/1.4 NA).
3
Multicolor Immunofluorescence Staining of Spleen
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