Megaview 3 camera

At 2 h, 24 h and 48 h post-treatment, AT samples were collected and fixed with a 2.5% glutaraldehyde and 2% paraformaldehyde in PBS for 2 h at 4 °C. The samples were then rinsed with PBS, post-fixed with 1% OsO₄ and 1.5% K₄Fe(CN)₆ for 2 h at 4 °C, dehydrated in acetone and embedded in Epon resin. Ultrathin sections were stained with Reynolds lead citrate and observed in a Philips Morgagni TEM (FEI Company) equipped with a Megaview III camera. At least three AT samples per animal were analysed.
The area of small lipid droplets extruding from the central one was measured in a total of 500 µm² of cytoplasm per experimental condition (X7′100) using the ImageJ software (NIH), and their total area was calculated and expressed as percentage of the measured cytoplasmic area.
A morphometric analysis was carried out also on 30 randomly-chosen mitochondria (X28′000) per control, O₂-, 10 µg- or 20 µg O₃-treated samples: the mitochondrial area and the ratio between inner and outer membrane (estimating the extension of cristae independently of the mitochondrial size) were assessed. The means ± SE were calculated and a statistical comparison was performed as described below.

Hundred micrometer samples of aSyn were incubated in PBS pH 7.4 alone, or in the presence of equimolar 4554W at 37°C, with agitation with a flea for 1 week. 5μL of each sample was mounted onto a carbon-coated copper grid for 2 min. Excess was blotted away, and grids stained with 4% uranyl acetate for 30 s. Images were collected on a 120 kV Tecnai G2 Spirit BioTWIN electron microscope (FEI) with a SIS Megaview III camera. Image and fibril length analysis was carried out using ImageJ and OriginLab, with the scale bar on each image used as the length reference. Four images were used for each analysis with number of measurements included in each analysis shown in Supplementary Table 2.