Durcupan resin

For ultrastructural analysis using Transmission electron microscopy (TEM), kidneys and podocyte cell cultures were fixed in 4% PFA and 2% glutaraldehyde (#4157, Carl Roth, Karlsruhe, Germany) in a 0.1 M cacodylate buffer (#11650, Science Services, München, Germany). The samples were postfixed in 1% osmium tetroxide (Science Services, #E19150) in double-distilled water (ddH₂O) and then washed six times in ddH₂O. The tissue was stained en bloc in 1% uranyl acetate solution (Science Services, #E22400-1) and washed two times in ddH₂O. Dehydration was performed via sequential incubation steps in 30%, 50%, 70%, 90%, and 2 × 100% ethanol (#32205, Fisher Scientific, Hampton, NH, USA) and then 2× 100% acetone (#179124, Sigma-Aldrich, St. Louis, MO, USA). All incubation steps were microwave-assisted (BioWave Pro+, PELCO, Fresno, CA, USA). After embedding in Durcupan resin (Sigma-Aldrich, #44611 and #44612), ultrathin sections (55 nm) were performed using a UC7 ultramicrotome (Leica), collected on Formvar-coated (Science Services, #E15830-25) copper grids (#G2500C, Plano GmbH, Wetzler, Germany). Post-staining was conducted for 1 min with 3% lead citrate (#11300, Delta Microscopies, Mauressac, France), and imaging was performed using a Talos L120c TEM (ThermoFisher, Waltham, MA, USA).

EM was performed as previously described (Costello et al., 2017b (link)). In brief, monolayers of cells were fixed for a total of 1 h in 0.5% glutaraldehyde in 0.2 M Pipes (pH 7.2) and postfixed in 1% osmium tetroxide (reduced with 1.5% wt/vol potassium ferrocyanide) in cacodylate buffer for 1 h. After washing in deionized water, the cells were dehydrated in a graded ethanol series before embedding in Durcupan resin (Sigma-Aldrich). 60-nm ultrathin sections were collected on pioloform-coated 100-mesh copper EM grids (Agar Scientific) and contrasted with lead citrate before imaging with a JEOL JEM 1400 transmission electron microscope operated at 120 kV. Images were taken with a digital camera (ES 1000W CCD; Gatan). Quantification of peroxisome–ER contacts was performed as previously (Costello et al., 2017b (link)). In brief, peroxisomes were sampled (n = 36–55, mean = 44 ± 1 [Fig. 4] or n = 106–116, mean = 112 ± 1.46 [Fig. 7] peroxisomes per experimental grid) by scanning the EM grids systematic uniform random. To estimate the mean fraction of total peroxisome membrane surface in direct contact with the ER, a stereologic approach by line intersection counting was used. Intersections were classified as direct membrane contact (defined as “attachment”) if there was <15-nm distance between peroxisome and ER membranes.

Three Sprague–Dawley male rats (∼6-week-old) of ∼150 g were used. After terminal anaesthesia was induced by brief inhalation of isoflurane (0.05% in air), followed by an intramuscular injection of ketamine (100 mg kg⁻¹) and xylazine (10 mg kg⁻¹), rats were intracardially perfused with 4% PFA and 0.1% glutaraldehyde in PBS (0.1 M, pH 7.2), and brain sections (100 μm) were cut on a Leica VT1000S vibratome (Leica Microsystems)19 (link)60 (link). The sections were immunolabelled with rabbit anti-Rph3a polyclonal antibody (1:100; ab68857, Abcam), followed by a biotinylated secondary antibody, ABC Elite Kit (Vector Laboratories), and the peroxidase reaction was revealed by ImmPACT VIP substrate Kit (Vector Laboratories). Then the sections were osmicated, dehydrated and flat embedded in Durcupan resin (Sigma-Aldrich). Ultrathin sections (70–90 nm) were countercoloured with uranyl acetate and lead citrate. Control experiments, in which the primary antibody was omitted, resulted in no immunoreactivity. The sections were visualized with a Philips CM100 transmission electron microscope (FEI) at 100 kV. The images were captured with an AMT XR40 4 megapixel side mounted CCD camera at a magnification between × 7,900 and × 92,000.

Adult heads were decapitated, halved, fixed for 30 min in 2.5% glutaraldehyde + 1% O_SO₄ in 0.1 M sodium phosphate buffer pH 7.2 for 30 min on ice, and incubated in 2% O_SO₄ in 0.1 M sodium phosphate buffer pH 7.2 for 2h on ice. Retinas were then dehydrated with successive 10 min incubations on ice in 30%, 50%, 70%, 90%, and twice 100% EtOH. The tissue was incubated 3x 10 min on ice in propylene oxide, placed in 50% propylene oxide/50% soft Durcupan resin (Sigma) for 12h at room temperature, and then in 100% resin for 4h at room temperature. Retinas were then transferred to BEEM flat embedding molds (Ted Pella) and baked at 70°C for 12h, cut into 2 μm sections with a microtome, and mounted in DPX (Sigma). Samples were imaged at 100x under phase contrast.

Six well plates with surfaces covered with ACLAR^® film (Electron Microscopy Sciences, PA, USA) were plated with cells at a density of 6 × 10⁵ cells per plate, and exposed to 40 µg/ml O-GNR-PEG-DSPE for 5 min or 12 h. At the end of two time points, cells were fixed with 2.5% electron microscopy grade glutaraldehyde (Electron Microscopy Sciences) in 0.1 M PBS. After fixation, the films containing fixed cells were placed in 2% osmium tetroxide in 0.1 M PBS, dehydrated through graded ethanol washes and embedded in durcupan resin (Sigma-aldrich). Areas with high cell densities were blocked, cut into 80 nm ultrathin sections using an Ultracut E microtome (Reichert-Jung, Cambridge, UK), and placed on formvar-coated copper grids. The sections were then viewed with a Tecnai Bio Twin G transmission electron microscope (FEI), at 80 kV. Digital images were acquired using an XR-60 CCD digital camera system (AMT)