Entellan neu

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).

Slice cultures were fixed in paraformaldehyde (4%, 0.1 M phosphate buffer; Applichem, Darmstadt, Germany) and rinsed in 0.1 M phosphate-buffered salt solution (PBS). Thereafter, slice cultures were exposed for 20 min to toluidine-blue working solution, which was a mixture of 5 ml stock solution (1 g of Toluidine Blue O in 100 ml of 70% ethanol; Sigma-Aldrich) and 45 ml of 1% NaCl solution (pH 2.0–2.5). Thereafter, 96% ethanol (100 ml of 96% ethanol and 4 drops of acetic acid) was used for color-differentiation of the staining. The differentiation step with strong acid removes unspecific staining of weak acidic structures and, thus, increases the contrast between background and stained cells. The process was stopped using 0.1 M PBS, once the differentiation was clearly visible. After brief rinsing with double distilled water, slice cultures were placed on object plates and dried overnight. The slices were then exposed to xylol (Sigma-Aldrich) for 10 min and embedded with Entellan Neu (Merck Millipore, Schwalbach, Germany).

To study the tissue morphology of 5 dpf ca10a and ca10b morphant larvae a histochemical analysis was performed. Prior to the analysis the larvae were washed with PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 3 hours at room temperature and the fixed larvae were transferred to 70% ethanol and stored at 4°C before being embedded in paraffin. The paraffin embedded samples were sectioned into 5 μm slices for the histochemical staining.
The fixed sections were deparaffinized in xylene, rehydrated in an alcohol series and histologically stained with Mayer's Hematoxylin and Eosin Y (both from Sigma-Aldrich). After dehydration, the slides were mounted with Entellan Neu (Merck; Darmstadt, Germany), examined and photographed using a Nikon Microphot microscope (Nikon Microphot-FXA, Japan).
To detect apoptotic cell death in the ca10a and ca10b morphants and the control larvae, a TdT-UTP nick end labeling (TUNEL) assay was performed for the prepared slides using the QIA39 FragEL DNA Fragmentation Detection Kit (Merck Chemicals Ltd., Nottingham United Kingdom). Briefly, the deparaffinized sections of the larvae were incubated with the TdT enzyme followed by incubation with anti-digoxigenin. Fluorescence staining was detected and photographed using a Nikon Microphot microscope (Nikon Microphot-FXA, Japan).