Preparation of fresh-frozen dried ultrathin sections by drying without vacuum.

Abstract

Fresh-frozen dried ultrathin sections were prepared without fixation, embedding or staining. Small blocks of tissues were chilled by making contact with copper blocks precooled with liquid nitrogen according to a procedure modified from that of Christensen. Sections were cut at -95 degrees C with a Reichert Um 2 ultramicrotome fitted with cryoaccessories, using dry glass knives. The sections were dried for 15 min or more on formvar-coated grids by a flow of dry nitrogen gas at -95 degrees C in a newly devised glass chamber placed in the cryokit compartment. The sections showed only a few holes attributable to ice crystal formation. Holes were found in the intercellular spaces as well as within cells. The sections were observed without staining and gave enough contrast to permit recognition of organelles such as nucleus, perinuclear cisternae, nucleolus, interchromatin granules, perichromatinic granules and secretory granules in the pancreatic exocrine cells. In the basal part of the proximal convoluted tubules of mouse kidney, abundant mitochondria were seen. Mitochondrial cristae and lines corresponding to the basal infoldings of the renal convoluted tubules were less dense than their surroundings. This method can yield ultrathin sections lending themselves to investigations of the distribution of elements with electron-probe X-ray microanalyzer and of diffusible substances by dry-mount autoradiography at the electron-microscopic level.