NCMIR methods for 3D EM: a new protocol for preparation of biological specimens for serial block face scanning electron microscopy

Abstract

Note: This protocol was designed to enhance signal for backscatter electron imaging of epoxy embedded mammalian tissue at low accelerating voltages (1-3 keV). However, it can easily be adapted for use with tissues from other species, tissue culture cells, plants and microbial cells by adjusting the buffer strength and the duration of relevant steps. This combinatorial heavy metal staining protocol employs a battery of contrasting steps after primary aldehyde fixation including: ferrocyanide reduced osmium tetroxide postfixation, thiocarbohydrazide-osmium liganding (OTO) and subsequent uranyl acetate and en bloc lead aspartate staining. Calcium chloride is included in a number of steps to enhance membrane preservation and staining. This protocol was designed primarily to emphasize the contrast of membranes. Many other contrasting agents may be included to increase staining of other cellular and extracellular constituents.