The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters

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Abstract
The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper. Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the ‘whorls’ observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative. Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid, in a molar ratio 7∶2∶1. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K4[Fe(CN)6] containing OsO4 in the post-fixation step we were able to demonstrate an increase in the visualization of membranous structures (liposomes).