Embed812 epoxy resin

Mouse duodenums were fixed in a mixed aldehyde fixative in cacodylate buffer. Tissues were contrasted with osmium tetroxide and uranyl acetate, dehydrated in an ascending series of alcohols, transitioned in propylene oxide and embedded in EmBed812 epoxy resin (Electron Microscopy Sciences). Embedded tissues were sectioned to glass slides and stained with toluidine blue to aid in final tissue selection. Selected areas were sectioned at ~80 nm and sections picked up on copper parallel bar grids. Samples were examined on a Thermo Fisher Scientific F20 transmission electron microscope operating at 80 kV and imaged with an AMT side-mount camera system.

Primary hippocampal neurons were grown on 3 mm carbon-coated sapphire disks until DIV 25, then fixed using 2% PFA with 2% glutaraldehyde in 0.1 M cacodylate buffer. Cells were postfixed with 1% osmium tetroxide, followed by 1% uranyl acetate. The samples were then dehydrated using an ethanol gradient, prior to infiltration with EmBed 812 epoxy resin (Electron Microscopy Sciences) using BDMA as an accelerator. Resin-filled BEEM capsules were attached to the cell surface to enable separation of the cell layer from the disk, and the resin was cured at 60°C for 36 h. Ultrathin sections (90 nm) were then cut by diamond knife using a Leica UC6 Ultramicrotome, and mounted on formvar-coated copper slot grids for viewing the ultrastructure.

Adult flies were anesthetized with CO₂ and heads were cut into half to ensure proper penetration of the fixative. The samples were put into freshly made fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde and 0.05% Triton X-100 in 0.1M sodium cacodylate buffer (pH 7.2) on rotator for at least 4 hours until all fly eyes were sunk to the bottom of the tube, then change to same fixative without Triton and continue fixed at 4°C for 4 days on rotator. After washing, the fly eyes were post fixed in 1% OsO₄ for 1.5 hour, dehydrated in a series of ethanol solutions (30%, 50%, 70%, 85%, 95%, 100%), followed by two rinses with propylene oxide and embedded in EMbed812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA). 500nm thick semi-thin sections were cut, mounted on glass slide and baked on hot plate overnight at 60°C. The sections are stained with 0.1% Toluidine blue, dried on hot plate and cover-slipped with Permount mounting medium (Electron Microscopy Sciences, Hatfield, PA) for light microscopy. 70nm ultra-thin sections were cut and mounted on formvar coated slot grids and stained with uranyl acetate and lead citrate. Imaging was performed by an electron microscope (CM12, FEI, Eindhoven, The Netherlands) at 120 kV, and recorded digitally using a camera system (Gatan 4k x 2.7K) with software Digital Micrograph (Gatan Inc., Pleasanton, CA).

Swiss 3T3 cells (4 × 10⁵ per dish) were seeded in a 60-mm dish, and then serum-starved for 16 h. Streptavidin-coated iron oxide nanoparticles (final concentration 2 nM) and biotin-PDGF-BB (final concentration 14 nM) were mixed in serum-free DMEM at 37 °C for 10 min. Cells were stimulated by incubating in the PDGF/nanoparticle-containing DMEM for the indicated times. Cells were then rinsed with PBS and fixed in fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde at room temperature (RT), then scraped and pelleted into microtubes, and consecutively fixed overnight at 4 °C, Cells were post fixed in 1% OsO₄, dehydrated in a series of ethanol solutions (30%, 50%, 70%, 85%, 95%, 100%), and embedded in EMbed812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA). 70 nm ultrathin sections were cut, mounted on copper grids and stained with uranyl acetate and lead citrate by standard methods. Grids were viewed using a Philips CM12 TEM (Philips) transmission electron microscope and photographed with Gatan 4k x 2.7k digital camera (Gatan Inc.).

The cell morphology of bacterial cells from the late log phase was examined using TEM analysis using JEOL JEM-1010 (JEOL, Akishima, Japan). The PHA granules formation by bacterial isolate was analyzed as per the methods described by Sandoval et al. (2007 (link)) with slight modification. The bacterial cells were grown to stationary phase in 2.0% glucose and harvested at 2,400 g, washed three times with phosphate buffer saline, and fixed by glutaraldehyde solution (2.0%, v/v) at 4°C. Samples were washed three times with PBS buffer and fixed with 1.0% osmium tetroxide for 60 min in the dark. The washing step was repeated, stained in blocks with uranyl acetate (30%), and embedded slowly in Embed 812 epoxy resin (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Polymerization of blocks was carried out in an oven at 50°C for 72 h. Ultrathin sections of the samples were cut using an ultramicrotome, and further grids were prepared. Structural studies were performed with an FEI Technai G2200kV transmission electron microscope (FEI, Hillsboro, Oregon, USA).