Epon 812 resin

Mice were injected IP or IV with 0.8 mg of F-AuNSs suspended in 100 μL PBS . The mice were sacrificed at 7-, 45- or 90-days postinjection. Liver, spleen, and kidneys were then retrieved. All tissues were dissected into 1 mm by 1 mm pieces with razor blades and immediately immersed in 2.5% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.4 ± 0.1) for 24 h at 4 °C. The specimens were further fixed with 1% osmium tetroxide for 1 h and enblock stained with 1% uranyl acetate for 30 min. The samples were dehydrated with a graded ethanol series and then infiltrated with Epon 812 resin (Electron Microscopy Sciences) using propylene oxide as a transition solvent. The Epon 812 was polymerized at 60 °C for 48 h, sectioned at 70 nm using a Leica EM UC7 Ultramicrotome and collected on 300 mesh nickel grids. The sections were stained with 2% uranyl acetate and lead citrate. TEM imaging was performed on a Tecnai T12 at 100 kV using an AMT CCD camera. The surface area of >100 single particles in TEM images at each time point was measured with ImageJ. The average surface area of those particles at each time point was compared that to as-prepared F-AuNSs before injection.

Flat block molds (Electron Microscopy Sciences) were half filled with Epon-812 resin and heated at 60°C until the resin reached a tacky consistency (approximately 4 hours). Samples were then placed in blocks, the brains oriented anterior up and dorsal forward (toward the block face). Blocks were filled with resin and allowed to fully polymerize for 48 hours at 60°C. After polymerization, thick sections (500nm) were cut using a Leica UCT Ultracut microtome until the dorsal most brain region appeared (first sight of brain tissue in sections). Thick section (500nm) cutting was then continued until the MB calyx was reached (approximately 40μm). Thin sections (65nm) were then cut for another 20μm, and collected on formvar-coated grids (Electron Microscopy Sciences) at 10 sections per grid. Images were acquired using a Philips CM10 transmission electron microscope (TEM) at 80kV. Measurements were taken with ImageJ freehand selection tool. 5–10 PN boutons were quantified within each MB calyx. To eliminate the chance of reimaging synaptic boutons, only one section per grid was imaged, with at least 5 grids (~3μm) of space between each grid examined. 3 animals were used for each EM grid, with 5 MB calyces analyzed per genotype. For sample size, 16 control and 22 dfmr1 sections were examined, with 70 control and 80 dfmr1 boutons, respectively.