Axio vert a1 fluorescence microscope

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).

Indirect-IF was performed to detect EHV-4 specific viral antigen in cell culture. Briefly, ED cells were grown in a 24-well plate and infected with 50 plaque forming units (PFU) of the isolated EHV-4 viruses. After 48 h (hrs) post infection, cells were fixed with 4% paraformaldehyde (PFA) and incubated for 30 min at room temperature (RT). Fixed cells were permeabilized with 0.1% Triton X-100 for 5 min and blocked with 3% bovine serum albumin (BSA; VWR^® life science; Radnor, PA, USA) for 1 hr. Cells were incubated overnight with primary EHV-4 anti-glycoprotein D monoclonal antibody (kindly provided by Dr Jules Minke, Merial, Lyon, France; dilution: 1:400) at 4 °C [35 (link)]. After washing, cells were probed with goat anti-mouse IgG (H + L) labeled with Alexa fluor-568 (A11019, Invitrogen^®, Carlsbad, CA, USA; dilution: 1:500) for 1 hr. Mock infected cells were stained with the same dilutions of primary and secondary antibodies. Plates were analyzed using Zeiss^® Axio Vert.A1 fluorescence microscope (Carl Zeiss AG, Jena, Germany).

The AGS cells were cultured at a density of 10 × 10³ cells per well in the culture medium described above on 8-well Nunc^® Lab-Tek^® II chamber slides covered with CC2 glass slide coverslips. After 2 days, the medium was renewed, and the cells were cultured in serum-free medium (300 μL) for 24 h. For GPR39-siRNA depleted cells, the AGS cells were transfected using Lipofectamine 2000 (Invitrogen; CA, US). Serum-starved cells were stimulated or not with obestatin (200 nM, 24 h) at 37°C. After 24 h, intact cells were fixed with 4% buffered paraformaldehyde-PBS for 30 min, washed, permeabilized with 0.25% Triton X-100 in PBS for 45 min, and blocked with PBST (1% Triton X-100, 1% Tween 20, 5% heat-inactivated normal goat serum, 1% BSA in PBS) for 30 min. For GPR39-siRNA depleted cells, the AGS cells were incubated with anti-GPR39 rabbit antibody diluted in 1% BSA in PBST (1 h, RT). After three washes with PBS, the cells were incubated with Phalloidin CruzFluor 594 and goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor^® 488 in 1% BSA in PBST (1 h, RT). DAPI was used to counterstain the cell nuclei (Invitrogen). Digital images of cells were acquired with a Zeiss Axio Vert.A1 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

Cells were seeded at a density of 10,000 cells/cm² on glass coverslips pretreated with 30 μg/mL poly-L lysine to promote adherence. At the end of the treatment cells were fixed with 4% paraformaldehyde (Euroclone) for 10 min at 4 °C and permeabilized for 10 min at RT with 0.1% (v/v) Triton X-100 (Bio-Rad). After washing, cells were incubated, according to dilution suggested by manufacturer, with anti-Tau (D1M9X) anti-Phospho-Tau (Thr231) or anti-β-Tubulin (D3U1W) (all from Cell Signaling Technology), for additional 45 min. After washing with PBS, cells were incubated for 30 min at RT with Alexa Fluor 594/488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Controls were performed by omitting the primary antibody. Slides were mounted with ProLong Gold antifade mounting medium with DAPI (Cell Signaling Technology). Finally, cells were observed with Zeiss Axio Vert. A1 fluorescence microscope (Zeiss, Jena, Germany) and acquired images were digitally elaborated with a modular image-processing and analysis software (Zen 2012 SP2 Blue Edition).