Osmium tetroxide post-fixation and periodic acid-Schiff dual-staining technique to demonstrate intracellular lipid and glycogen in the mouse liver section – a novel method for co-visualization of intracellular contents in paraffin-embedded tissue

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Intracellular accumulation of excessive glycogen or lipid in the liver occurs as a manifestation of metabolic dysfunction or drug-induced toxicity in humans or animals. The identification of individual components (lipids, glycogen, or water) involved is essential to the diagnosis of disease and pathogenesis. The aqueous fixatives and processing solvents used in paraffin embedding for routine hematoxylin and eosin stains remove both lipid and glycogen, leaving clear vacuoles of which the precise content is unknown in routine hematoxylin and eosin-stained sections. Traditional methods for detecting intracellular lipids and glycogen require two separate techniques, such as periodic acid-Schiff (PAS) reaction, with or without diastase digestion for glycogen, and osmium tetroxide (OsO4) post-fixation for lipid and utilization of different tissue sections. We describe here, for the first time, a novel OsO4/PAS dual-staining technique that permits a simple color-coded detection and direct visualization of both lipids and glycogen in the same paraffin-embedded tissue section. In this study, liver samples were collected from diet-induced obese (C57BL/6) or db/db mice and fixed in 10% neutral buffered formalin for 24 h. The formalin-fixed samples were post-fixed with OsO4, processed, sectioned, stained with PAS reaction, and evaluated microscopically for lipid and glycogen contents. Both lipid and glycogen were clearly identifiable with subjectively quantifiable severity and distribution in the same liver section. In conclusion, OsO4/PAS dual-staining, in our opinion, could be used in routine practice and provides a resource-saving innovation in differentiating glycogen from lipids in the same section of paraffin-embedded tissue sections.

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