Anti mhcii

Mice ear tissues were cut into pieces and placed in 2.5 mg/ml Dispase II (Roche) solution at 4°C overnight. Then the epidermis was torn off and digested with 0.025% trypsin at 37°C for 15 min, and an equal volume of FBS was added. After being filtered through a 70-μm cell strainer, the cells were resuspended in PBS with 1% FBS. For detecting Tregs, mice ear tissues were cut into pieces and placed in the digestion cocktail (a solution consisting of 2 mg/ml collagenase XI [Cat# C9407; Sigma-Aldrich], 0.5 mg/ml hyaluronidase [Cat# H3506; Sigma-Aldrich], and 0.1 mg/ml DNase [Cat# D5025; Sigma-Aldrich]) with shaking at 255 rpm in 37°C for 40 min (Moreau et al., 2021 (link)). Then an equal volume of FBS was added. After being filtered through a 70-μm cell strainer, the cells were resuspended in PBS with 1% FBS. Dead cells were excluded using Fixable Viability Dye eFluor 780 (eBioscience). The single-cell suspensions were stained per the manufacturer’s recommendations with anti-CD207 (Cat# 144206; BioLegend), anti-MHC II (Cat# 107605; BioLegend), anti-CD3ε (Cat# 152311; BioLegend), anti-γδ TCR (Cat# 118107; BioLegend), anti-CD3 (Cat# 152311; BioLegend), anti-CD4 (Cat# 100406; BioLegend), and anti-Foxp3 (Cat# 563101; BD Pharmingen). After washing with PBS, the cells were subjected to flow cytometry analysis (BD Fortessa II), and the data were analyzed using FlowJo v10.4.

Mice were perfused with PBS under physiologic pressure and aortas were harvested. Tissues were embedded in O.C.T. compound (Sakura Tissue Tek, Alphen aan den Rijn, the Netherlands) and sectioned in 6 μm thickness using Leica CM3050S cryostat. Hematoxylin and eosin, Verhoeff-Van-Gieson, and Masson trichrome stainings were conducted. Immunostaining was also performed according to standard protocols with the following antibodies: anti-CD206 (AF2535, R&D Systems, Minneapolis, Minn), anti-F4/80 (123120, Biolegend, San Diego, Calif), anti-MHCII (107616, Biolegend), anti-MYH11 (ab53219, Abcam, Cambridge, UK), and anti-IL-1β (AF-401, R&D Systems). A total of three sections, approximately 60 μm apart, were stained and analyzed for each mouse aorta. The integrated density (the sum of the value of the pixels in the image or selection), area of immunofluorescence (square pixels), and number of signal positive cells were measured using ImageJ Software (National Institutes of Health, Bethesda, Md), as previously described.¹⁶ (link)