Math5

Cells were washed with PBS, then fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. After two washes with PBS, cells were incubated with a blocking buffer (10% goat serum and 0.3% Triton X-100 in PBS) for 1 h. Primary antibodies were diluted in blocking buffer and then added to cells for 2 h at room temperature and then incubated with antibodies against Math5 (1:500; Millipore, Bethesda, MA, USA), Brn3b (1:250; Santa Cruz, Dallas, TX, USA), and Tuj1 (1:500; Abcam, Cambridge, UK). The cells were then subjected to three 3-minute washes with PBS and incubated with secondary antibodies diluted in a blocking buffer containing Hochest (1:10,000; Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. The following secondary antibodies were used: Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-rabbit IgG, Alexa 488 goat anti-mouse IgG, and Alexa 594 goat anti-mouse IgG (Thermo Scientific, Waltham, MA, USA). After three 3-minute washes with PBS, the cells were visualized using confocal microscopy (Zeiss LSM 700, Zeiss, Jena, Germany).

For immunofluorescent staining, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After two washes with PBS, the cells were incubated with blocking buffer (10% goat serum and 0.3% Triton X-100 in PBS) for 1 h. Primary antibodies were diluted in blocking buffer and then added to the cells for 2 h at room temperature. The following primary antibodies were used: Bestrophin, MITF, PMEL17, AMPA R2 and Tuj1 (Abcam), ZO-1 (Thermo Fisher Scientific), Math5 (Millipore) and Brn3a (Santa Cruz). The cells were then subjected to three 3-min washes with PBS and incubated with secondary antibodies Alexa Fluor 488 or Alexa Fluor 594 of goat anti-rabbit or goat anti-mouse IgG (Thermo Fisher Scientific) diluted in blocking buffer containing DAPI (Thermo Fisher Scientific) for 1 h at room temperature. After three 3-min washes with PBS, the cells were visualized using confocal microscopy (Zeiss LSM 700).

Live cells were cultured in MitoTracker Red (Cell Signaling, Danvers, MA, USA) at 37 °C for 30 min and then with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min. Next, cells were incubated with blocking buffer containing 10% goat serum and 0.3% Triton X-100 in PBS for 1 h and then incubated with primary antibodies against Brn3b (1:250; Santa Cruz, Dallas, TX, USA), Math5 (1:500; Millipore, Bethesda, MA, USA), and Thy1 (1:500; Abcam, Cambridge, UK) at 4 °C overnight. The cells were then washed with PBS and incubated with secondary antibodies diluted in a blocking buffer containing Hoechst (1:10,000; Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. The following secondary antibodies include sheep and Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-rabbit IgG, Alexa 488 goat anti-mouse IgG (Thermo Scientific, Waltham, MA, USA), and Alexa 594 goat anti-mouse IgG (Thermo Scientific, Waltham, MA, USA). Fluorescent staining of mitochondria is then performed using the MitoTracker Red CMXRos (Cell Signaling, Danvers, MA, USA). Finally, the cells were detected by confocal microscopy (Zeiss LSM 700, Zeiss, Jena, Germany).