Neurofilament h nfh

For ICC, the cells (5 × 10⁴ cells/mL) were cultured on poly-L-lysine-coated aclar plastic coverslips as described previously [29 (link)]. Medium containing 1% FBS (EMD Millipore Corp.) was changed to medium containing the HDAC inhibitors. The cells were fixed with 4% paraformaldehyde (PFA; T&I Co., Chuncheon-si, Republic of Korea) for 15 min at room temperature. The cells were then blocked with 0.5% Triton X-100 (Sigma–Aldrich) for 20 min and 10% normal goat serum (NGS; Vector Laboratories, Inc., Burlingame, USA) for 30 min. Primary antibodies were added for 1.5 h, and the secondary antibodies were then added and kept in the dark for 1 h. For staining nuclei, 4′,6-diamidino-2-phenylindole (DAPI,  1 μg/mL, Life Technologies Corporation) was added for 30 min. The cells were then imaged with a Zeiss LSM510 confocal microscope (Carl Zeiss, Jena, Germany) after mounting [29 (link)]. The primary antibodies used were microtubule-associated protein 2 (MAP2, 1:200; Cell Signaling Technology, Danvers, USA) and neurofilament-H (NFH, 1:400; Cell Signaling Technology). Alexa 488-conjugated goat anti-rabbit (Invitrogen Co., Waltham, USA) and Alexa 594-conjugated goat anti-mouse (Invitrogen Co.) were used as the secondary antibodies. All experiments were repeated at least three times.