Uniform Serial Sectioning for Transmission Electron Microscopy

Preparation of serial thin sections from a specific region of interest. a, Stimulating electrodes positioned in a hippocampal slice. a', Diagram of a region of interest surrounding the indentations left by the stimulating electrodes on the slice, which is embedded in agarose for stability. b, Fixed region of interest illustrated diagrammatically in b' and turned on edge to obtain slices using a vibrating blade microtome to produce “vibra-slices” as illustrated in c'. c, The 70-μm-section vibra-slices are gently transferred into a 24-well tissue culture plate containing 0.1 m phosphate buffer using a small brush at the corner of the surrounding agarose to avoid mechanically induced dark artifacts in the tissue. d, On-edge view of a vibra-slice through the depth of the hippocampal slice from the air to the net surface; this vibra-slice is through the indentation left by the stimulating electrode (stim). e, Neighboring vibra-slice used to obtain series near the stim, by shaving off the Epon to the dotted line. The scale bar in e is for d and e. f, Toluidine blue-stained thick section at the bottom is used to guide where to place the trapezoid on the face of the Epon block. The goal is to have a long ribbon of uniform section thickness. g, Uniform ribbon sectioning, except where the section arm was stopped and restarted. h, Routine Lowicryl embedded tissue does not ribbon well. i, After hair spray, the same trapezoid sections well.