Evos fl auto epifluorescence microscope

After 120 h of incubation, cell-seeded scaffolds (crosslinked and laminin-coated scaffold cross-sections) were removed from the incubator. Cells were then fixed with paraformaldehyde 4% (w/v) for 20 min at room temperature and permeabilized with 0.5% (v/v) Triton^TM X-100 (Millipore-Sigma) in PBS 1× for 10 min. Samples were further incubated with NGS 10% (v/v) in PBS 1× for 60 min at 37 °C to block non-specific sites. Samples were then washed with PBS 1× and incubated with primary Rabbit monoclonal antibodies against S100β (E7C3A) (#90393, diluted 1:800, Cell Signaling Technology Inc., Danvers, MA, USA) overnight at 4 °C under gentle agitation. Following 3 washes with PBS 1×, samples were further incubated with Anti-rabbit IgG (H + L), F(ab′)2 Fragment Alexa Fluor^® 488 Conjugate (#4412, diluted 1:500, Cell Signaling Technology Inc.) and Hoechst 33342 for nucleus counterstaining (5 µg/mL) for 90 min at 37 °C in a humidified incubator (5% CO₂) under gentle agitation. Stained cells were kept in fresh PBS 1× and visualized at magnifications of 4×, 10×, and 40× with an EVOS FL Auto epifluorescence microscope (Life Technologies, Carlsbad, CA, USA).

Promastigote cells were incubated for 72 h without RAD or with the IC₉₀ of RAD. After the incubation period, cells (1 × 10⁷ cells) were sedimented, washed twice with PBS and suspended in 1 ml of PBS. Aliquots of the suspension (2 × 10⁵ cells) were applied on microscopic slides and air-dried. After fixing the cells for 2 min in ice-cold methanol, the slides were air-dried for 20 min. Non-adherent cells were removed by gentle washing (0.1% Triton X-100 in PBS) followed by incubation in blocking solution (2% BSA, 0.1% Triton-X 100 in PBS). Slides were then incubated for 1 h with monoclonal mouse anti-tubulin (Sigma-Aldrich, München, Germany, 1:4000), washed thrice and then incubated for 1 h with anti-mouse Alexa 594 (Dianova, Hamburg, Germany, 1:250) and DAPI (Sigma-Aldrich, München, Germany, 1:25). After washing the slides thrice, Mowiol and coverslips were applied and the slides were left to dry for 24 h at 4 °C. Fluorescence microscopy was carried out on an EVOS FL Auto epifluorescence microscope (Life Technologies, Darmstadt, Germany).

MPG were harvested after sacrifice (by CO₂) and fixed for 20 hours in 4%PFA, transferred to 30% sucrose for 48 hours, and then cryosectioned in 10 μm slices longitudinally. Sections were collected sequentially onto slides. Per MPG, 2–3 sections, 100–150 μm apart from each other, were used for quantification of nNOS⁺ somas. Sections were immunostained for nNOS (Santa Cruz Biotechnology; SC-5302) and tyrosine hydroxylase (Abcam; ab112) (as a counterstain to visualize sympathetic neurons) as well as DAPI according to standard IHC protocol. Slides were imaged on an EVOS FL Auto epifluorescence microscope (Life Technologies, Thermo Fisher Scientific) using a ×10 objective. nNOS⁺ somas in the caudal region of the MPG proximal to the CN were quantified (areas ranged 0.25–0.5 mm²). If there was a tear in the tissue in the analyzed region, this area was subtracted from the total area. Eight to 10 sections from 3–4 MPG were analyzed. Imaging and analysis for these experiments were performed in a blinded manner.