Lr white resin

The collected B16-F10 cells were stored overnight at 4 °C in a fixative solution containing 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2. Fixed cells were washed in cacodylate buffer and post-fixed with 2% (v/v) osmium tetroxide in 0,1 M cacodylate buffer, pH 7.2 for 2 h at 4 °C. Samples were washed in the same buffer and dehydrated through an ascending series of ethanol and embedded in LRWhite resin (Electron Microscopy Science, PA, USA). For ultrastructural observations at least 20 ultra-thin sections (60–90 nm) were obtained using a Reichert Ultracut ultramicrotome equipped with a diamond knife (Leica Microsystems). Ultra-thin sections were collected on copper grids, stained with uranyl acetate and lead citrate, and observed with a 1200 EXII electron microscope (Jeol, Tokyo, Japan). Micrographs were captured by the SIS VELETA CCD camera equipped with iTEM software (Olympus, Tokyo, Japan).

For direct NP internalization, LAD2 cells were treated for 3 and 24 h at 37 °C and 5% CO₂ with (1/100 v/v) PLGA/PVA-21, PLGA/PVA-22, or PLGA/PVA; untreated cells were included as controls. LAD2-treated cells were collected, centrifuged at 230× g, and fixed overnight with 2% paraformaldehyde/2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1M phosphate buffer pH 7.2 (Sigma-Aldrich CAN, Oakville, ON, Canada). Fixed LAD2 cells were immersed in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) at room temperature for 1 h before the sequential dehydration with ethanol of 50%, 70%, 95%, and 100%. The dehydrated cells were embedded in LR white resin (Electron Microscopy Sciences, Hatfield, PA, USA), and cured at 55 °C for 12 h. The ultrathin sections with 100 nm thickness, sliced by an ultra-microtome, were placed on formvar-coated copper TEM grids double-stained with uranium acetate and lead citrate. Bright field TEM images of LAD2 cell thin sections were obtained on JEOL2200FS TEM.

Array tomography analyses were performed as previously described (Hudry et al, 2013 (link); Kay et al, 2013 (link)). Five to six pieces of cortical tissue (1 mm³) were dissected and fixed for 3 h in 4% paraformaldehyde and 2.5% sucrose in 0.01 M PBS. After dehydration in ethanol, the samples were incubated in LR White resin (Electron Microscopy Sciences) overnight at 4°C before polymerization at 53°C. Ribbons of 20–40 ultrathin (70 nm thick) serial sections were cut with a Histo Jumbo diamond knife (Diatome) on an ultracut microtome (Leica) and mounted on glass coverslips.

To visualize Drosophila retina ultrastructure, adult fly heads were dissected, fixed, dehydrated, and embedded in LR White resin (Electron Microscopy Sciences) as described [67 (link)]. Thin sections (80 nm) prepared at a depth of 30–40 μm were stained with uranyl acetate and lead citrate (Ted Pella) and examined using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a Gatan CCD (4k × 3.7k pixels, USA).
TEM of photoreceptor terminals was performed as described [73 (link)]. Adult fly heads were dissected in 4% PFA and the retinas were removed. The dissected lamina was fixed in a solution with 4% PFA and 2.5% glutaraldehyde for 2 h on ice, followed by fixation in 1% osmium tetroxide for 1.5 h at 4°C. Tissues were then dehydrated in a series of ethanol dilutions at 4°C (10-min wash in 10, 25, 40, 55, 70, 85, 95, and 30-min wash in 100% ethanol for 5 times). Samples were gradually infiltrated with 2 ratios of ethanol and Eponate 12 (Ted Pella), finally going into 3 changes of pure resin. Samples were allowed to infiltrate in pure resin overnight on a rotator and embedded in Eponate 12 resin (Ted Pella). Thin sections (80 nm) were stained with uranyl acetate and lead-citrate (Ted Pella) and examined using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a Gatan CCD (4k × 3.7k pixels, USA).