Xt system benchmark autostainer

The ear and back skin tissues of the mice were removed, fixed with 10% formaldehyde, embedded in paraffin, and serially sectioned into 4–6-μm sections using a microtome (Leica Biosystems, Newcastle, United Kingdom). The tissues were mounted on slides and stained with hematoxylin/eosin solution (Sigma-Aldrich) or toluidine blue (Toluidine Blue Stain Kit, VitroVivo Biotech, Rockville, MA, United States). For immunohistochemical staining, the slide-mounted tissues were incubated overnight at 4°C with CD3 primary antibodies (1:150 dilution; cat.no. ab16669, Abcam, Cambridge, United Kingdom) and processed using a XT System Benchmark autostainer (Ventana Medical System, Tucson, AZ, United States) according to the manufacturer’s instruction. The cells positive for CD3 and toluidine blue staining as measured from the tissue sections were analyzed using Solution for Automatic Bio-Image Analysis software (Ebiogen, Seoul, South Korea).

The mouse tissue samples were fixed in 10% formalin. For tissue staining, the samples were embedded in paraffin and cut into 4-μm-thick sections. The sections were stained with hematoxylin/eosin solution (H&E) (Sigma-Aldrich, St. Louis, MO, USA) or Toluidine Blue (TB) O solution (Sigma-Aldrich). For immunohistochemistry, the sections were incubated overnight at 4 °C with F4/80 antibodies and processed using an XT System BenchMark autostainer (Ventana Medical System, Tucson, AZ, USA) according to the manufacturer’s instructions. The stained tissue samples were observed using a digital slide scanner (Kfbio, Ningbo, China). The tissue sections were analyzed using Solution for Automatic Bio-Image Analysis software (EBIOGEN, Seoul, Korea).