The quantitative retention of cholesterol in mouse liver prepared for electron microscopy by fixation in a digitonin-containing aldehyde solution

Abstract

A major methodological problem in the intracellular localization of cholesterol is the nearly complete extraction of sterols during routine dehydration and embedding procedures for electron microscopy. Cholesterol digitonide (a sterol complex with digitonin), however, is qualitatively insoluble in these solvents. Mouse liver has been prepared as follows: (a) Flickinger's aldehyde fixative, 20 hr; (b) Flickinger's fixative containing 0.2% digitonin, 24 hr; (c) cacodylate wash, 24 hr; (d) 1% OsO(4), 2 hr; (e) acetone dehydration; and (f) Epon 812 infiltration under vacuum, 28 hr. After the last step, an analysis of the tissue for sterol content under optimal analytical conditions demonstrates a retention of 99% of the unesterified cholesterol present in unfixed mouse liver. Liver prepared in an identical manner except for omission of digitonin is essentially devoid of sterols. Cholesterol isolated chromatographically from liver processed as outlined above has been identified unequivocally by mass spectrometry. Liver from step (f) also has been polymerized, thin-sectioned, and examined in the electron microscope. A remarkable quality of fine-structural preservation is obtained. The major alteration encountered is the presence of small cylindrical "spicules," often occurring as tightly packed concentric lamellae, at membrane surfaces.