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2 protocols using 2 2 2 tribromoethyl alcohol

1

Dendritic Cell Immune Response Assay

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Todd Hewitt broth, yeast extract, trypticase soy broth (TSB) and BactoTM agar were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Blood agar plates were purchased from Hanil Komed (Seongnam, Korea). 2,2,2-Tribromoethyl alcohol, 2-methyl-2-butanol, Red Blood Cell Lysis Buffer, and hematoxylin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Goat anti-mouse IgG Fc-, IgG1-, IgG2b-, and IgA-HRP were purchased from Southern-Biotech (Birmingham, AL, USA). A Mouse Dendritic Cell Enrichment Set was purchased from BD Biosciences (San Diego, CA, USA). The following antibodies were purchased from BioLegend (San Diego, CA, USA): APC-labeled anti-mouse IFN-γ, APC-labeled anti-mouse IL-4, PE-labeled anti-mouse IL-17A, PE-labeled anti-mouse CD3, APC-labeled anti-mouse CD8, PerCP-labeled anti-mouse CD11c, FITC-labeled anti-mouse I-Ab for MHC class II, APC-labeled anti-mouse CXCR5, PerCP-labeled anti-mouse B220, PE-labeled anti-mouse CD69, PE-labeled anti-mouse PD-1, PE-labeled anti-mouse CD80, and FITC-labeled anti-mouse CD86 antibodies. FITC-labeled anti-mouse CD4 antibody was obtained from BD Biosciences. All of the isotypes matched with each antibody were purchased from BioLegend or BD Biosciences.
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2

Viral Transduction and Perfusion Protocol

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After the final test, each animal, anesthetized with 2.5% Avertin (prepared by mixing 10 g of 2,2,2-tribromoethyl alcohol [T4,840-2; Sigma-Aldrich] with 10 mL of tert-amyl alcohol [24,048-6; Sigma-Aldrich], diluted 1:40 by phosphate-buffered saline and filter sterilization), received an intracranial infusion of Chicago Sky Blue (C8679; Sigma-Aldrich) (0.2%), followed by transcardial perfusion with 4% paraformaldehyde. Fluorescent and visible light microscopy identified the sites of viral transduction and cannulation.
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