Immunohistochemistry and Immunofluorescence staining of corneas

The collected corneas were fixed in 10% formaldehyde solution (Shanghai Merck Life Sciences Technology Co., Ltd, Sigma-Aldrich) for 48 h and decalcified with 5% nitric acid (Shanghai Merck Life Sciences Technology Co., Ltd, Sigma-Aldrich). After dehydration and washing, corneas were embedded in paraffin. For each specimen, cut consecutive sections parallel to the injured corneal region using a microtome. Immunohistochemical (IHC) staining was performed to evaluate the level of TGF-β, α-SMA, and MMP9 with corresponding antibodies. For immunofluorescence (IFC) staining, 10 μm thick frozen corneal samples were fixed with cold acetone (Shanghai Merck Life Sciences Technology Co., Ltd, Sigma-Aldrich) for 5 min, washed with cold PBS, and sealed with 2% donkey serum (Shanghai Merck Life Sciences Technology Co., Ltd, Sigma-Aldrich) at room temperature for 1 h. Then, slices were incubated with rabbit/mouse TGF-β antibody, α-SMA antibody, MMP9 antibody, Keratin 3 antibody, and Keratin 12 antibody overnight at 4 °C. After washing three times with cold PBS, slices were then stained with Alaxa488-labeled Donkey-anti-rabbit/mouse antibody for 1 h. After three rounds of washing with cold PBS, all slides were mounted with a mounting medium with DAPI (Shanghai Santa Cruz Biotechnology Co., Ltd) and covered with cover slides for imaging using a confocal microscope (Fig. 5b, e, h, j and Supplementary Fig. ²²). Antibodies: Anti-TGF-β-APC (Biolegend, Clone TW7-16B4, Catalog: 141406, Dilution: 1:400, Mouse Antibody), Anti-α-SMA-antibody (Biolegend, Clone 1A4, Catalog: 614852, Dilution:1:500, Mouse Antibody), Anti-TGF-β-antibody (Abcam, ab215715, Dilution: 1:500, Rabbit Antibody), Anti-α-SMA-antibody (Abcam, ab5694, Dilution: 1:200, Rabbit Antibody), Anti-MMP9-antibody (Abcam, ab283575, Dilution: 1:500, Mouse/Rabbit Antibody), Anti-Keratin 3-antibody (Abcam, ab77869, Dilution: 1:200, Mouse/Rabbit Antibody), Anti-Keratin 12-antibody (Abcam, ab185627, Dilution: 1:1000, Mouse/Rabbit Antibody).