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 cd3 alexafluor 647
Manufactured by BioLegend
Anti-CD3-AlexaFluor 647 is a fluorescently-labeled antibody that binds to the CD3 antigen expressed on T cells. It is designed for use in flow cytometry applications to identify and quantify T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti b220 brilliant violet 421, by BioLegend (1 mentions)
Rabbit anti laminin 1 2, by Abcam (1 mentions)
Lsm 710 axioobserver, by Zeiss (1 mentions)
Volocity v6, by PerkinElmer (1 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti cd56 pe, by Beckman Coulter (1 mentions)
Anti cd20 pe, by BD (1 mentions)
Anti cd19 fitc, by BioLegend (1 mentions)
Anti cd14 alexafluor647, by BioLegend (1 mentions)
Anti cd16 pe, by BioLegend (1 mentions)
Anti cd8 pe, by BD (1 mentions)
Opera phenix, by PerkinElmer (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
2 protocols using anti cd3 alexafluor 647
1
Immunofluorescent Tissue Staining and Imaging
2
Multiparametric Immunofluorescence Imaging
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For immunohistochemistry staining, all primary antibodies were diluted 1:300 in PBS with 6 µM DAPI (Sigma-Aldrich) for nuclear detection. Antibodies used were anti-CD3 Alexa Fluor 647 (BioLegend, Clone UCHT1), anti-CD4 FITC (BioLegend, clone SK3), anti-CD8 PE (BD Biosciences, clone SK1), anti-CD19 FITC (BioLegend, clone SJ25C1), anti-CD56 PE (Beckman Coulter, clone N901), anti-CD16 PE (BioLegend, clone 3G8), anti-CD14 Alexa Fluor 647 (BioLegend, clone HCD14) and anti-CD20 PE (BD Biosciences, clone 2H7). Per well, 20 μl of the antibody cocktail was added and incubated for 1 h at room temperature. For imaging, a PerkinElmer Opera Phenix automated spinning-disk confocal microscope was used and each well of a 348-well plate was imaged at 20× magnification with 5 × 5 non-overlapping images, covering the whole well surface. The images were taken sequentially from the bright-field (650–760 nm), DAPI/nuclear signal (435–480 nm), GFP signal (500–550 nm), PE signal (570–630 nm) and APC signal (650–760 nm) channels. Raw .tiff files were exported for analysis.
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