Mouse anti nfh mab

SH-SY5Y and DPSCs were seeded at a density of 2 × 10⁴ cells/mL in 6-well plates and standard culture medium for 24 h. After seeding, both cell lines were stimulated for 48 h and 10 days with DPSC-CM, as reported in Table 2 and incubated in conditions of hypoxia and normoxia for 5 and 16 days. At the end of the treatment, the obtained samples were used for immunofluorescence analysis. Briefly, SH-SY5Y and DPSCs treated as above were fixed with 4% paraformaldehyde for 10 min at 4 °C and permeabilized with permeabilizing solution (0.1% Triton X-100 in PBS) for 10 min at 4 °C. After washing in PBS, cells were incubated with mouse Anti-β3-tubulin mAb and mouse Anti-NFH mAb (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by Anti-mouse IgG Alexa fluor 488 (ThermoFisher Scientific, Rockford, IL, USA) in the dark for an additional 60 min. Finally, cells were observed with a Zeiss Axio Vert. A1 fluorescence microscope (Zeiss, Milan, Italy). Quantitative analysis of the fluorescence intensity for each protein of interest was measured by Image J.

hDPSCs were seeded (2 × 10⁴ cells/mL) 6-well plates, in DMEM-L containing serum and antibiotics. Twenty-four hours after seeding, hDPSCs untreated or treated with siRNA PrP or scrambled for 72 h were stimulated with recPrP^C for 14 days and tested for immunofluorescence analysis. Briefly, hDPSCs untreated or treated as above were fixed with 4% paraformaldehyde and permeabilized by 0.1% (v/v) Triton X-100. After washing, cells were incubated with mouse anti-B3-Tubulin mAb, mouse anti-NFH mAb (Cell Signaling Technology Danvers, MA, USA) and mouse anti-GAP43 mAb (Sigma-Aldrich, Milan, Italy) for 1 h at 4 °C, followed by anti-mouse alexa fluor 488 or anti-mouse alexa fluor 594 (Cell Signaling Technology Danvers, MA, USA) for additional 30 min. Finally, cells were observed with a Zeiss Axio Vert. A1 fluorescence microscope (Zeiss, Oberkochen, Germany).

Flow cytometry was used to quantify neuronal antigen expression on hDPSCs after a long period of stimulation with recPrP^C. Briefly, hDPSCs untreated or treated with siRNA PrP or scrambled siRNA for 72 h, were stimulated with recPrP^C for 14 days as described below and fixed with 4% paraformaldehyde and permeabilized by 0.1% (v/v) Triton X-100. After washing, cells were incubated with mouse anti-B3-Tubulin mAb, mouse anti-NFH mAb (Cell Signaling Technology Danvers, MA, USA) and mouse anti-GAP43 mAb (Sigma-Aldrich, Milan, Italy) for 1 h at 4 °C, followed by PE-conjugated anti-mouse IgG H&L (Abcam, Cambridge, MA, USA) for additional 30 min. All samples were analyzed with a FACScan cytometer (BD Accuri C6 Flow cytometer) equipped with a blue laser (488 nm) and a red laser (640 nm). At least 20,000 events were acquired.