B220 pe ra3 6b2

Spleens from immunized and control IL-27p28–eGFP transgenic mice were removed 6 h after polyIC administration and immediately fixed overnight using PLP buffer, as previously described (30 (link)). Spleens were flash frozen with liquid nitrogen in OCT and 20-μm tissue sections were obtained using a cryostat. Sections were left to dry for 30 min, blocked with 5% BSA for 1 h, and stained with anti-eGFP (Alexa Fluor 488 Rabbit polyclonal; Life Technologies), XCR1 (allophycocyanin clone ZET; BioLegend), and B220 (PE RA3-6B2; BioLegend) in 5% BSA. After washing extensively, sections were coverslipped with Fluoromount mounting medium and left to dry overnight. Slides were imaged on a Zeiss LSM 780 confocal microscope, and image analysis was performed with Fiji (31 (link)).

Mouse intrahepatic immunocytes were isolated as previously described (Liang et al., 2018 (link)). The isolated immunocytes were incubated with fluorescently conjugated antibodies directed against mouse CD45 (FITC, I3/2.3, Cat#: 147709), B220 (PE, RA3-6B2, Cat#: 103207), CD3e (APC, 145-2C11, Cat#: 100312), TCRβ (FITC, clone: H57-597, Cat#: 109205), and TCRγδ (APC, clone: GL3, Cat#: 118115) from Biolegend (San Diego, CA, United States) for 20 min at 4°C in the dark. B cells (CD45⁺ and B220⁺), γδT cells (CD3e⁺ and TCRγδ⁺), and αβT cells (CD3e⁺ and TCRβ⁺) were obtained from intrahepatic immunocytes by flow cytometry. For cytokine detection, the selected cells were stained with antibodies against murine tumor necrosis factor α (TNF-α) and interleukin-17 (IL-17) from BD PharMingen (Mountain View, CA, United States) for 20 min at 4°C in the dark after incubation with permeabilization buffer. The stained cells were examined on a Beckman CytoFlex S (Beckman Coulter, Inc., Kraemer Boulevard Brea, CA, United States) and analyzed with the FlowJo software (version 10.0, TreeStar, Ashland, OR, United States).