Bielschowsky

For staining of neurofibrils with Bielschowsky (Bio-Optica, Milan, Italy), NPC and cells differentiated over approaches A and B for 28 days were seeded onto poly-L-ornithine/laminin (Sigma-Aldrich, St. Louis, Missouri, USA) precoated Nunc™ Lab-Tek™ II Chamber Slides™ (Thermo Fisher, Waltham, Massachusetts, USA), fixed with 4% w/v PFA in PBS by incubation for 20 min at 4°C and washed twice with PBS. According to the manufacturer's protocol, the slide was washed twice with ultrapure water and was incubated with 10 drops of Reagent A for 15 min at 40°C. After washing two times with ultrapure water, 10 drops of Reagent B were added following incubation for 20 min at 50°C. The supernatant was discarded, and the slide was treated with reduction solution (20 drops Reagent C, 8 drops each of Reagents D, E, and F in 50 mL ultrapure water) for 2 min. Cells were washed twice with ultrapure water and subsequently incubated with 10 drops of Reagent G for 3 min. Before dehydrating the slide with ascending concentration of ethanol and treating twice with Xylene (Sigma-Aldrich, St. Louis, Missouri, USA), it was washed two times with ultrapure water. Finally, the slide was embedded in Entellan Neu (Merck, Darmstadt, Germany) following incubation for 30 min at room temperature to dry. The stained neuronal structures were imaged with Nikon Eclipse TS100 microscope (Nikon, Minato, Japan).