Electron Microscopy
Tissue was collected from formalin-fixed brain
and underwent fixation in 1% osmium tetroxide
and dehydration in increasing concentrations of
ethanol. The sample was then embedded in Epon.
Semithin sections (1.4 μm) were made, stained
with Azur II, and analyzed by means of light
microscopy. Ultrathin sections (60 nm) were
stained with uranyl acetate and lead citrate. In
addition, a small piece of brain (5 mm3) was
homogenized in buffer. The suspension was then
cleared by low-speed centrifugation, and the obtained
supernatant was ultracentrifuged directly
onto an electron microscopic grid with the use
of an Airfuge (Beckman Coulter). Negative staining
was performed with 1% phosphotungsticacid. Imaging of the ultrathin sections and brain
homogenate was performed with the use of a
120-kV JEM-1400Plus transmission electron microscope
(JEOL).