Agar 100 resin kit

Samples for TEM study were fixed with 4% glutaraldehyde in cacodylate buffer for 24 h at 4 °C and postfixed in buffered 1% osmium tetra oxide for 2 h at 4 °C, washed with cacodylate buffer 2–3 times, and kept overnight at 4 °C in cacodylate buffer before en bloc staining with 4% uranyl acetate (Agar Scientific Ltd R1043, Essex, England) in double distilled water for 10 min. Samples were then dehydrated in ascending series of ethanol and embedded in Epon (Agar 100 resin kit; Agar Scientific Ltd R1043).
Semi-thin 1 µm sections were obtained using a Leica ultramicrotome (Reichert Ultracuts; Leica Microscystem, Vienna, Austria) and stained with toluidine blue for 1–2 min before viewing with light microscopy. Ultrathin sections were cut at 70–80 nm, collected on copper grids (300 meshes), and stained with uranyl acetate and lead citrate (Agar Scientific). Images were viewed using TEM (Leo Libra 120; Carl Zeiss SMT AG, Oberkochen, Germany).

The cultures were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde. The cells were then rinsed with 0.15 M sodium cacodylate (pH 7.2–7.4) for 10 min and incubated in fresh 1% osmium tetraoxide in 0.1 M sodium cacodylate for 1 h at RT. After incubation, the sodium cacodylate was rinsed away to dehydrate the dishes with 70% ethanol for 30 min, 95% ethanol for 30 min and > 99% ethanol for 1 h. A thin plastic layer (Agar 100 resin kit, Agar Scientific Ltd) was added to the dishes and incubated for 1 h. The plastic was poured off and a new plastic layer was added onto the dishes for incubation overnight in a desiccator. Next, the plastic was heated to enable its removal after which a new thicker plastic layer was added before another incubation for 1 h in a desiccator. Cells were covered with 3-mm plastic and polymerized in the oven at 60 °C for 48 h. Embedded cells were sectioned by using a Leica ultracut UTC ultramicrotome (Rowaco AB) and visualized with a Tecnai G2 transmission electron microscope (FEI company) with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

All general chemicals were from Sigma (St. Louis, MO, USA). Reagents for cell culture and Alexa Fluor® 488 - conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). TrueBlack® was from Biotium (Fremont, CA, USA). Goat anti-human Cathepsin D antibody was from Santa Cruz Biotechnology (Dallas, TX, USA), mouse anti-bovine RPE65 antibody and rabbit anti-human GFAP were from Abcam (Cambridge, UK). Ketamine, xylazine, tropicamide and oxybuprocaine chlorhydrate were from Centravet (Maison-Alfort, France). Lubrithal eye gel was from Dechra Pharmaceuticals (Northwich, UK). Optimal cutting temperature compound and other reagents for histology were from Roth Sochiel (Lauterbourg, France). Agar 100 resin kit was from Agar Scientific (Stansted, UK). 9'-cis-Norbixin was prepared from 9'-cis-bixin (AICABIX P, purity 92 %) purchased from Aica-Color (Cusco, Peru) upon alkaline hydrolysis as previously described [22 (link)] and according to Santos et al. [64 (link)]. The obtained product (the 9'-cis isomer) showed an HPLC purity of 97 % as confirmed by ¹H-nuclear magnetic resonance (using malonic acid as internal standard). Fresh solutions of 9'-cis-norbixin, stored as powder at -80°C, were prepared in DMSO.

The neuronal cultures were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde. The cells were then rinsed with 0.15 M sodium cacodylate (pH 7.2–7.4) for 10 min and incubated in fresh 1% osmium tetraoxide in 0.1 M sodium cacodylate for 1 h at RT. After incubation, the sodium cacodylate was rinsed away to dehydrate the dishes with 70% ethanol for 30 min, 95% ethanol for 30 min and > 99% ethanol for 1 h. A thin plastic layer (Agar 100 resin kit, Agar Scientific Ltd) was added to the dishes and incubated for 1 h. The plastic was poured off and a new plastic layer was added onto the dishes for incubation overnight in a desiccator. Next, the plastic was heated to enable its removal after which a new thicker plastic layer was added before another incubation for 1 h in a desiccator. Cells were covered with 3 mm plastic and polymerized in the oven at 60 °C for 48 h. Embedded cells were sectioned by using a Leica ultracut UTC ultrotome (Rowaco AB) and visualized with a Tecnai G2 transmission electron microscope (FEI company) with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

Cells were briefly washed in PBS prior to fixation in 2.5% glutaraldehyde (1 h or overnight at 4C). The dishes containing the cells were rinsed with 0.15 M sodium cacodylate buffer (pH 7.2–7.4) for 10 min, and incubated in newly prepared 1% osmium tetraoxide in 0.1 M sodium cacodylate for 1 h at RT. Sodium cacodylate was rinsed away to dehydrate the dishes with 70% ethanol for 30 min, 95% ethanol for 30 min and >99% ethanol for 1 h. Following dehydration, a thin newly made plastic layer (Agar 100 resin kit, Agar Scientific Ltd) was added to the dishes and incubated for 1 h to permit evaporation of the alcohol. The plastic was poured off and a new plastic layer was added onto the dishes for incubation O/N in a desiccator. Next, the plastic was heated to enable its removal and a new thicker plastic layer was added and dishes were incubated for 1 h in a desiccator. Cells were covered with a ∼3 mm plastic and polymerized in oven at 60C for 48 h. By using a Leica ultracut UTC ultrotome (Rowaco AB), embedded cells were sectioned and studied in a Tecnai G2 transmission electron microscope (FEI Company) with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

For room-temperature transmission electron microscopy (TEM), cells were fixed for 2 h with a pre-warmed solution comprising 2.5% glutaraldehyde and 1.4% sucrose (pH 7.2); then, they were processed for epoxy resin embedding, as detailed by the manufacturer (Agar100 Resin Kit, Agar Scientific, Essex, UK) and as described in previous work from this group [21 (link)]. Sectioning was performed on a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany), and the ultra-thin sections (40–60 nm) were mounted on carbon and Formvar-coated 50-mesh copper grids and counterstained with 1% uranyl acetate and Reynolds lead citrate (Agar Scientific, Essex, UK). Imaging was done using a 200 kV Talos F200C TEM, equipped with a 4K × 4K Ceta camera (Thermo Fisher Scientific, Waltham, MA, USA).