S7113

Freshly isolated type II cells were plated on a plastic chamber slide (Lab-Tek II chamber slides, Nalge Nunc International, Naperville, IL). After overnight culture, the cells were infected with adenovirus (Ad-lacZ or Ad-Rab³8) at MOI = 2 to reduce expression level of Rab³8 protein and facilitate identification of co-localization with SP-B. The cells were further cultured for 24 h and washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 10 min and followed with 100% acetone for 30 s. The fixed cells were blocked with 25% normal goat serum, incubated with a primary antibody (anti-Rab³8 and anti-SP-B), followed by a secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG antibody and Alexa 594-conjugated goat anti-mouse IgG antibody) (Abcam, Tokyo, Japan). The slides were mounted with an antifade solution containing DAPI (S7113, Millipore, Temecula, CA). The immunofluorescence images were acquired using a fluorescence microscope (Keyence Biorevo BX-9000, Osaka, Japan).

For the differentiation NSCs using HDACi alone, the cells were treated with freshly prepared 1 mM NaB in distilled water (Cat# B5887, Sigma Aldrich, St. Louis, USA). NaB treatment was performed at 2 h after NSCs passage as single cell culture. Neural differentiation of NSCs was assessed by flow cytometry and immunocytochemistry on day-7 after NaB treatment.
For flow cytometry, the cells were fixed with 4% paraformaldehyde for 20 min at 4 °C. Subsequently, the cells were incubated with MAP-2 specific primary antibody (1:1000, cat # ab5392, Abcam, Cambridge, UK) at room temperature. After one hour, the cells were washed with phosphate buffer saline (PBS) and the primary antigen-antibody reaction was detected by incubating the cells for 1 h at room temperature with fluorescently conjugated secondary antibody diluted in 5% goat serum. The MAP-2 positive cells were analyzed by flow cytometry (BD Bioscience, San Jose, USA). For microscopic analysis of MAP-2 positive cells, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; Millipore S7113, Billerica, USA) and observed using fluorescence microscope (Olympus BX53; Tokyo, Japan). The MAP-2 positive neurons were counted in various microscopic fields and compared with the untreated control group.