1400 tem

Animals were anesthetized with an intraperitoneal injection of a drug cocktail containing ketamine (100 mg/kg) and xylazine (10 mg/kg) diluted in a 0.9% NaCl solution. After anesthesia, mice were sacrificed with decapitation. Brains were quickly dissected and fixed in 0.1% Glutaraldehyde, 4% PFA, 2% Sucrose in 0.1 M Sorensen Phosphate Buffer for 24 h. The area of interest, caudoputamen, was dissected and fixed again overnight in 2.5% Glutaraldehyde, 0.1% Sucrose in 0.1 M Sorensen Phosphate Buffer. Samples were then washed in PBS and post‐fixed in 2% osmium tetroxide in PBS. After dehydrating the samples in a graded series of isopropanol, they were blockstained in 2% uranyl acetate in ethanol and embedded in Epon. Semi‐thin (500 nm) sections were stained with toluidine blue and analyzed using light microscopy to find the area of interest. Ultra‐thin sections (80 nm) were cut on a microtome using a diamond knife (Leica EM UC7) and collected on carbon‐coated formwar films on 200 mesh copper grids (Plano) contrasted with 0.3% lead citrate for 1 min and imaged using a TEM 1400 (Jeol). For the experiments, only axons localized in axon fiber bundles passing through the caudoputamen were chosen. For the quantification of non‐myelinated axons, only axons with caliber over 200 nm were chosen. Analysis of the images was performed with ImageJ software.

Cells were cultured with BN NP and HAP for different time periods (8, 12, and 24 hrs) according to the experimental conditions. They were then treated in a step-by-step manner (Buffer wash, post fixation, dehydration, and resin implantation). The cultured cells were fixed using 3% Glutaraldehyde (10 min) and rinsed twice using 0.1 M Cacodylate buffer (20 min). Then they were treated with 1% Osmium Tetroxide for an hour. They were rinsed again with UHQ water thrice (10 min each) before being treated with 1% Uranyl Acetate for an hour. Cells were then dehydrated by multiple exchange of ethanol, using progressively higher ethanol concentration starting from 50% to 100%. This was followed by resin (LX112) implantation in the culture dish. The concentration of the resin was varied from a low to high resin ethanol ratio (1:2, 1:1, 2:1). Finally the cells were treated with 100% percent resin (1 h) before being placed in the oven to be moulded at 70 °C (24 hrs).
The processed samples were then sectioned in 80 nm thickness using the Leica UC7 Ultramicrotome. The sectioned samples were placed in a 400 mesh carbon grids and taken to JEOL TEM 1400 for imaging. Samples were then carefully placed in the TEM holder and secured; making sure they would not be displaced inside the microscope.

Based on nitrogen sorption measurements on a Quadrasorb SI (Quantachrome) the BET surface area and NL-DFT (equilibrium kernel for silica with cylindrical pores) pore size distribution of the particles was calculated. Particle morphology and particle size was visualized by transmission electron microscopy on a TEM 1400 (Jeol). Dynamic light scattering was used to measure the hydrodynamic diameter as well as the zeta potential of the particles in a dispersion of particles (0.1 mg/mL) in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer solution (25 mM, pH 7.2) from Merck KGaA (Darmstadt, Germany) on a Zetasizer Nano-ZS ZEN 3600 (Malvern Instruments).