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Strains were grown statically from 1x10⁷ conidia at 37°C in MM for 24 h. Mycelia were harvested and immediately fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 2.5% (v/v) of glutaraldehyde and 2% (w/v) of paraformaldehyde for 24 h at 4°C. Samples were encapsulated in agar (2% w/v) and subjected to fixation (1% OsO₄), contrasting (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with Spurr resin (Electron Microscopy Sciences) of 16 h and 3 h at RT. Additional infiltration was provided under vacuum at RT before embedment in BEEM capsules (Electron Microscopy Sciences) and polymerization at 60°C for 72 h. Semithin (0.5-μm) survey sections were stained with toluidine blue to identify the areas of best cell density. Ultrathin sections (60 nm) were prepared and stained again with uranyl acetate (1%) and lead citrate (2%). Transmission electron microscopy (TEM) images were obtained using a Philips CM-120 electron microscope at an acceleration voltage of 120 kV using a MegaView3 camera and iTEM 5.0 software (Olympus Soft Imaging Solutions GmbH). Cell wall thickness of 100 sections of different germlings were measured at 23,500 x magnification and images analyzed with the ImageJ software [77 (link)]. Statistical differences were evaluated by using test-T.

Whole sections of mouse eyes were thoroughly investigated by electron microscopy. Total numbers of eyes investigated by electron microscopy for the different strains were 12 eyes (pigmented Abca4^−∕−), 16 eyes (albino Abca4^−∕−), 16 eyes (Abca4^−∕−.Rdh8^−∕−), six eyes (albino WT) and three eyes (pigmented WT). Sections were examined for changes in photoreceptor, RPE and Bruch’s membrane structure. Lipid droplets in RPE cells were counted and their diameter measured in whole sections. Thickness of Bruch’s membrane was measured in 15 consecutive images. All measurements were performed with iTEM 5.0 Software (Olympus Soft Imaging Solutions, Muenster; Germany).

Strains were grown statically from 1 × 10⁷ conidia at 37°C in MM for 24 h. Mycelia were harvested and immediately fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 2.5% (vol/vol) glutaraldehyde and 2% (wt/vol) paraformaldehyde for 24 h at 4°C. Samples were encapsulated in agar (2%, wt/vol) and subjected to fixation (1% OsO₄), contrasting (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with Spurr resin (Electron Microscopy Sciences) of 16 h and 3 h at RT. Additional infiltration was provided under vacuum at RT before embedment in BEEM capsules (Electron Microscopy Sciences) and polymerization at 60°C for 72 h. Semithin (0.5-μm) survey sections were stained with toluidine blue to identify the areas of best cell density. Ultrathin sections (60 nm) were prepared and stained again with uranyl acetate (1%) and lead citrate (2%). Transmission electron microscopy (TEM) images were obtained using a Philips CM-120 electron microscope at an acceleration voltage of 120 kV using a MegaView3 camera and iTEM 5.0 software (Olympus Soft Imaging Solutions GmbH). Cell wall thicknesses of 100 sections of different germlings were measured at ×23,500 magnification, and images were analyzed with the ImageJ software (85 (link)). Statistical differences were evaluated by using one-way analysis of variance (ANOVA) and Tukey’s post hoc test.

The isolated extracellular vesicles were immediately fixed in 0.1M sodium phosphate buffer (pH 7.4) containing 2.5% (v/v) of glutaraldehyde and 2% (w/v) of paraformaldehyde for 2 h at 4°C. Samples were encapsulated in agar (2% w/v) and subjected to fixation (1% OsO4), contrasting (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with Spurr resin (Electron Microscopy Sciences) of 16 h and 3 h at RT. Additional infiltration was provided under vacuum at RT before embedment in BEEM capsules (Electron Microscopy Sciences) and polymerization at 60°C for 72 h. Semithin (0.5-μm) survey sections were stained with toluidine blue to identify the areas of best cell density. Ultrathin sections (60 nm) were prepared and stained again with uranyl acetate (1%) and lead citrate (2%). Transmission electron microscopy (TEM) images were obtained using a Philips CM-120 electron microscope at an acceleration voltage of 120 kV using a MegaView3 camera and iTEM 5.0 software (Olympus Soft Imaging Solutions GmbH).

Third instar larvae were dissected in fresh Ca²⁺ free HL3 (110 mM NaCl, 5 mM KCl, 10 mM NaHCO₃, 5 mM Hepes, 30 mM sucrose, 5 mM trehalose, and 10 mM MgCl₂, pH 7.2;^{87 (link)}), nerves were cut and larvae subsequently incubated for 10 min in HL3 with 1.5 mM CaCl₂ and 60 mM KCl. Multiple steps of washing with HL3 before fixation removed the non-internalized dye. Larvae were fixed in 4% paraformaldehyde (Laborimpex, 15714) and 2.5% glutaraldehyde (Polysciences, Inc, 111-30-8) in 0.1 M sodium-cacodylate buffer pH 7.4 (Merck, C0250-500g) at 4 °C for at least 24 h. The next day the larvae were osmicated in 2% osmium tetroxide (Laborimpex AGR1023) for 2 h on ice. After staining in 2% aqueous uranyl acetate solution (EMS #22400) for at least 1.5 h and dehydration in an ascending series of ethanol solutions, the samples were embedded in Agar 100 (Laborimpex, AGR1031) and cured at 60 °C for 48 h. Ultrathin sections (70 nm) were cut with a Dumont Diamond Knife on a Leica UCT ultra-microtome and collected on copper grids (Van Loenen, 01805-F) and imaged on a JEM 1400 transmission electron microscope (JEOL) at 80 kV with a bottom mounted camera (Quemasa; 11 megapixels; Olympus) running iTEM 5.2 software (Olympus). Statistical analyses were performed on larvae from 3 independent crosses per genotype. More than 5 NMJs per larvae were imaged.

Adult fly heads were dissected and immediately fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M Na-Cacodylate buffer (pH 7.4) for 2 h at room temperature. Samples were further fixed at 4 °C overnight, and then washed with 0.1 M Na-Cacodylate, pH 7.4, and subsequently osmicated with 2% osmium (OsO₄/Na-Cacodylate buffer). After dehydration in an ascending series of ethanol solutions and staining in 4% aqueous uranyl acetate solution the specimen were embedded in Agar 100 (Laborimpex; Agar Scientific). Ultrathin sections (70 nm) of the retina and the lamina were collected on grids (Laborimpex; Agar Scientific) coated with Butvar and imaged on a JEM 1400 transmission electron microscope (JEOL) at 80 kV with a bottom mounted camera (Quemasa; 11 megapixels; Olympus) running iTEM 5.2 software (Olympus).