Immunohistologic techniques for detecting the glycolipid Gb3 in the mouse kidney and nervous system

摘要

Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb3) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb3 in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb3 was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb3 using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb3 was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb3 localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb3 was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb3 in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.