Fa016 100g

Immunohistochemistry (IHC) staining was performed as described previously (Jiang et al., 2016 (link); Yi et al., 2021 (link)). In brief, the paraffin slices were soaked in xylene, 100% ethanol, 95% ethanol and 70% ethanol successively for dewaxing and hydration. After that, the slides were put into EDTA buffer (pH 9.0, MVS-0099, MXB Biotechnologies) and maintained at 100°C for 20 min to retrieve the antigen. The slides were blocked with blocking buffer (5% bovine serum albumin, FA016-100G, Genview) for 30 min at 37°C after treatment with 3% hydrogen peroxide for 40 min. The IRF9 primary antibody (Proteintech, 14167-1-AP, at 1:500 dilution) was added to the slides for incubation overnight at 4°C after removing the blocking solution. The peroxidase-conjugated secondary antibody (Kit-9902, MXB Biotechnologies) was incubated for 30 min at 37°C. A DAB kit (DAB-0031, MXB Biotechnologies) was used to develop the color, and the sections were counterstained with hematoxylin. The slices were mounted with neutral resins (10004160, Sinopharm) after dehydration and observed under the microscope. The intensity of medial IRF9 staining was quantified relative to the area of the medial layer to evaluate the relative expression level of IRF9.