Epon 812 resin

We collected tissue samples immediately after MRI; the left NAc of S1 and A1 mice was immersed in 2.5% PBS-buffered glutaldehyde at 4 °C and sent to the TEM laboratory of the Histology Core Facility at the MGH Center for Systems Biology for preparation and double-blinded examination. After tissue was dehydrated in ascending concentrations of ethanol, immersed in propylene oxide, and embedded in Epon 812 resin (Agar Scientific Ltd., Standstead, England), samples were cut into ultrathin sections (~60 nm). The Core prepared tissue with and without standard TEM stain using osmium tetroxide (1%, 2 h), uranyl acetate (Ua, 2%, 5 min) and Reynold’s lead citrate [20 (link)]. We found that standard staining masked NP identification; we modified TEM staining by omitting all stains, unless indicated, to reduce the background of membrane structure and enable visualization of SPION. The neuronal nucleus was identified as a smooth, round nuclear body with diameter of ~7 μm. We defined microglia (MG) by the presence of irregular euchromatic nucleus, a peripheral rim of heterochromatin, and various empty and partially filled lysosomes/exosomes (Ly/Ex).

Cells were fixed with a 25 mM sodium acetate/acetic acid buffer containing 2.5% glutaraldehyde, 0.05% cuprolinic blue (Electron Microscopy Sciences, Hatfield, PA, USA), and 0.05 M magnesium chloride overnight at room temperature, keeping Petri dishes on a rocking platform. Cells were then post-fixed with 2% osmium tetraoxide (Agar Scientific, Stansted, Essex, UK), dehydrated with graded ethanol solutions, and embedded into Epon 812 resin. Ultrathin sections were collected onto formvar-coated 2 × 1 mm slot copper grids and contrasted with uranyl acetate and lead citrate. Observations and photographic recording were made using a Philips CM12 STEM transmission electron microscope.