Ki67 sola15

Intestinal tissues were fixed with 1% (w/v) paraformaldehyde (Electron Microscopy Services) in PBS for 16 h, washed with PBS, and equilibrated in 30% (w/v) sucrose for a further 16 h. Tissues were then frozen at −80°C in Optimal Cutting Temperature (OCT) embedding medium (Thermo Fisher Scientific). Cryostat sections were cut at a thickness of 20-30 μm, air-dried for 1 h, then rehydrated for 10 min in PBS and blocked with a 0.1 M Tris solution containing 1% (w/v) mouse serum, 1% (w/v) bovine serum albumin (BSA), and 0.1% (w/v) Triton X-100 for 1 h at room temperature. Sections were stained overnight at 4°C with a combination of the following antibodies in blocking buffer at a 1:100 dilution: CD45.2 (104; Thermo Fisher Scientific), IgG (SouthernBiotech), and Ki67 (SolA15; Thermo Fisher Scientific). Additionally, actin was stained in certain experiments using Phalloidin dyes (Thermo Fisher Scientific) at a 1:200 dilution. Confocal imaging was carried out on a Leica SP8 confocal microscope. Images were analyzed using Imaris software (Bitplane).

Cell staining was performed with the appropriate antibody cocktail in FACS buffer. The cell subsets were identified based on following cell surface markers: AMs (CD11c⁺Siglec F⁺CD64⁺MerTK⁺), Neutrophils (CD11b⁺Ly6G⁺), BMDMs (F4/80⁺CD11b⁺), macrophage polarization marker (CD80, CD86, CD71 and CD206). Fluorescence-conjugated antibodies CD11b (M1/70), CD11c (N418), CD64 (X54-5/7.1), MerTK (2B10C42), Ly6G (1A8), F4/80 (BM8), CD86 (GL-1), CD80 (16-10A1), CD71 (RI7217) and CD206 (C068C2) were purchased from BioLegend; Ki67 (SolA15) was purchased from Invitrogen; Siglec F (E50-2440) was purchased from (BD Biosciences, Franklin Lakes, NJ, USA). For Ki67 staining, cell suspensions were stained for the surface marker at 4 °C for 30 min in the dark. Cells were washed twice with FACS buffer prior to fixation and permeabilization with the Foxp3 transcription factor staining buffer set (eBioscience, San Diego, CA, USA) for 1 h at RT in the dark. Cells were washed twice with perm wash buffer (eBioscience) and stained with Abs against Ki67 and control immunoglobulin (BioLegend) in perm wash buffer for at least 30 min at RT in the dark. Cells were washed twice with perm wash buffer before samples were processed with the flow cytometer. Samples were collected on flow cytometry and analyzed using FlowJo v10.6.2 software (Tree star) (BD Biosciences, Franklin Lakes, NJ, USA).

Single cell suspensions were stained with fixable viability dye eFluor^® 780 (eBioscience) for 30 min at 4°C, and Fc receptors were blocked with anti-CD16/32 (BioLegend) blocking antibody prior to surface staining with antibodies. Antibodies for surface staining used are listed, mouse: CD45 (30-F11, BioLegend), TCR β (H57-597, BioLegend), CD69 (H1.2F3, eBioscience), CD1d tetramer (NIH); human: CD45 (HI30, eBioscience), TCRα/β (IP26, BioLegend), CD69 (FN50, BioLegend), Vα24 (6B11, BD Bioscience). For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend). For Ki67 staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (eBioscience), and further stained with Ki67 (SolA15, eBioscience). Data were acquired using LSR II (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).