Ultracut ultramicrotome

Since compromised B-CNS-B integrity was determined in motor neuron areas in ALS mice at early and late disease stages (Garbuzova-Davis et al., 2007a (link), 2007b (link)), structural analyses of cervical and lumbar spinal cord microvessels were performed using electron microscopy. Briefly, spinal cord tissue samples were postfixed in 1% osmium tetroxide (Electron Microscopy Sciences, Inc., Hatfield, PA) in 0.1M PB for 1 hour at RT and then dehydrated in a graded series of acetone dilutions. Tissues were transferred to a 50:50 mix of acetone and LX112 epoxy resin embedding mix (Ladd Research Industries, Burlington, VT) and infiltrated with the mix for 1 hour. The tissues were then transferred to a 100% L×112 embedding mix and infiltrated with fresh changes of this embedding mix. The tissues were further infiltrated overnight in fresh embedding medium at 4ºC. On the following day, the tissues were embedded in a fresh change of resin in tissue capsules. The blocks were polymerized at 70°C in an oven overnight. The blocks were trimmed and then sectioned with a diamond knife on a Leica Ultracut ultramicrotome. Thin sections were cut at 80–90 nm, placed on copper grids, and stained with uranyl acetate and lead citrate.

For cross-section visualization using TEM, cells cultured on a nanopillar substrate underwent an extended overnight fixation in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) at 4°C. The sample was then washed in 0.1M sodium cacodylate buffer with 0.2 M sucrose, and post-fixed in osmium (1% osmium tetroxide with 0.8% potassium ferricyanide in 0.1 M sodium cacodylate), all at 4°C. After washing three times with ice-cold distilled water, the sample was en-bloc stained with 2% uranyl acetate on ice, washed three times again with ice-cold distilled water, gradually dehydrated in an ethanol series (Gold Shield) to 100% ethanol, and then exchanged to acetonitrile. The cell sample was infiltrated with epoxy resin (Embed 812) and hardened after baking at 65°C for 24 hrs. Then, the bottom quartz coverslip was etched away by 49% hydrofluoric acid (Avantor). The sample was then re-embedded with fresh pure resin to refill the spaces left by the coverslip. The resulting resin block was then trimmed and sectioned on a Leica Ultracut ultramicrotome into 70 nm-thick sections. The sections were collected on 1 × 2 mm copper slot grids with carbon-formvar support film (Electron Microscopy Sciences) and post-stained for 30s in 1:1 uranyl acetate:acetone. The grids were imaged at 120 kV on a JEOL 1400 TEM.