Immunohistochemistry

The collected cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Endogenous peroxidase activity was blocked with 3% (v/v) H₂O₂ for 5 min at room temperature. The slides were blocked in 5% bovine serum albumin (Hyclone, Thermo Fisher Scientific, Inc.) at room temperature for 2 h and incubated with a monoclonal anti-nestin primary antibody (1:200; cat no. OM264981; Omnimabs) overnight at 4°C, followed by incubation with a horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Immunohistochemical staining was visualized using 3,3′-diaminobenzidine chromogen for 3 min at room temperature. Images were captured using a routine light microscope (magnification: 200×; BX51; Olympus Corporation) and at ≥5 fields in each slide were selected at random.