All experiments were done with approval from the Weizmann Institute Animal Care and Use Committee. Animals were sacrificed at the age of 10 wk. Blood and pancreata samples were stained with AlexaFluor488 anti-CD45, AlexaFluor647 anti-NK1.1, and AlexaFluor647 anti-CD3 antibodies (Biolegend) and analyzed by FACS or ImageStram. The SA-β-gal and H&E stainings were performed as described (Krizhanovsky et al. 2008 (link)). To detect immune cells in tissue sections from the pancreas, the mice were subjected to a series of seven hourly intraperitoneal injections of Caerulein (50 ng/g per injection) for 2 d in a row to induce pancreatitis. Twenty-four hours after the last injection, mice were sacrificed, and pancreata were taken, fixed in 4% PFA for 5 h at room temperature, cryoprotected in 30% sucrose, and snap-frozen in OCT. Cryosections (10 µm) were then stained with the same antibodies as for FACS.
Alexafluor647 anti cd3
Manufactured by BioLegend
AlexaFluor647 anti-CD3 is a fluorescently-labeled antibody that specifically binds to the CD3 antigen expressed on the surface of T cells. It can be used for the detection and analysis of T cells in flow cytometry applications.
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2 protocols using alexafluor647 anti cd3
1
Immune Cell Profiling in Pancreatitis
2
Immunofluorescent Staining of Mouse Tissue
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Fresh mouse tissues were soaked in 4% paraformaldehyde (PFA) for 10 hours and then sectioned at a thickness of 100 μm by a vibrating blade microtome (Leica VT1200, Germany). Then, slices were blocked in PBS containing 0.02% Triton X-100 (Sigma, Missouri, USA) and 1% bovine serum albumin (BSA) for 1 hour and then incubated with a directly-labeled antibody overnight at 4 °C. Primary antibodies used in this study were as follows: PE anti-CX3CR1 (Clone SA011F11 Cat# 149005, BioLegend), Alexa Fluor 647 anti-CD11c (Clone N418 Cat# 117312, BioLegend), and Alexa Fluor 647 anti-CD3 (Clone 17A2 Cat# 100209, BioLegend). For indirectly-labeled antibody staining, the slices were blocked as described above, then incubated with goat anti-mouse VAChT (Clone ABN100, Sigma) for 1 hour at room temperature, and further stained with the secondary antibody donkey anti-goat IgG(H + L) (Clone A21447, Biolegend) overnight at 4 °C. After washing off the unbound antibody, samples were enclosed using PBS containing 50% glycerol (w/w). Samples were imaged under an inverted confocal fluorescence microscope (FV3000, Olympus, Japan) with dry 10× NA0.4 and dry 20× NA0.8 objectives.
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