Immunocytochemical Analysis of Keratocytes

The procedure for immunocytochemistry was performed following the previously reported protocols^{28 (link)}. Keratocytes were seeded at a density of 2 × 10⁴ cells per milliliter and grown on 4-well Lab-Tek chamber slides (Nalgene Nunc International, Penfield, NY, USA), and 0, 5, and 10 ng/mL of TGF-β1 were treated for 72 h. Cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature, and permeabilization was conducted using 0.1% triton x-100 for 5 min at RT. Following the washing steps with DPBS, cells were blocked using 1% bovine serum albumin (BSA) in DPBS for 30 min at room temperature. The chamber slides were incubated overnight at 4 °C with rabbit polyclonal anti-α smooth muscle actin (1:1000; catalog number: sc-53142; Santa Cruz, Biotechnology). The chamber slides were then washed with DPBS and incubated with Alexa488-conjugated donkey anti-mouse antibody (1:1000; catalog number: A21202; Molecular Probes) for 2 h at room temperature. Staining for F-actin was executed using tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (1 µg/mL; Sigma-Aldrich, St. Louis, MO, USA). The counterstaining of cell nuclei was carried out using 4′,6-diamidino-2′-phenylindole (DAPI, 10236276001; Roche Diagnostics GmbH, Mannheim, Germany) with mounting solution. Slides were viewed using a fluorescence microscope.