Following in vivo 2-photon imaging of an area of spinal cord with chronic experimental autoimmune encephalomyelitis, a correlated block-face SEM volume was collected of an area near blood vessels displaying clustering of glial cells (either oligodendrocytes, microglia, or both) or control blood vessels lacking clustering.
The animal was perfused with Ringer's solution followed by 0.5% glutaraldehyde / 2% PFA in cacodylate. The region of spinal cord under the imaging window was cut from the perfused cord and post-fixed for 2 hours in cold 0.5% glutaraldehyde / 2% PFA in cacodylate. The specimen was then post-fixed overnight in cold 4% PFA in cacodylate. The dorsal aspect of the cord was cut into 150 µm thick horizontal vibratome sections. The sections were post-fixed overnight in cold 2% glutaraldehyde in cacodylate overnight. The tissue was stained with 2% osmium tetroxide (Ted Pella) in 0.15M cacodylate, 0.5% aq. thiocarbohydrazide (Electron Microscopy Sciences), 2% aq. osmium tetroxide, 2% aq. uranyl acetate (Ted Pella), and lead aspartate, with thorough washing with water between each staining solution. The sections were then dehydrated through ethanol and acetone and then infiltrated with Durcupan ACM (Millipore Sigma). The sections flat-embedded between glass slides coated with mold-release compound (Electron Microscopy Sciences, Hatfield PA) and were cured at 60 °C for 72 hours. Specimens were imaged on a Zeiss Gemini 300 VP SEM equipped with a focal charge compensation system and a Gatan 2XP 3View system. Volumes were collected at 2.5 kV with 1 µsec dwell time, 10 nm pixels, 50 nm step size, and focal gas injection with nitrogen gas turned on. The scope was run in analytic mode and high current mode. The resulting stacks of images were aligned using a custom Python script using IMOD programs.