Immunohistologic techniques for detecting the glycolipid Gb(3) in the mouse kidney and nervous system.
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 (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) 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 Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) 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 Gb(3) 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 Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.
Kolling
Glynis L
GL
Department of Internal Medicine/Nephrology, University of Virginia, Charlottesville, VA 22908, USA.
Obata
Fumiko
F
Gross
Lisa K
LK
Obrig
Tom G
TG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D005629:Frozen Sections / Q000379:methods*; D006463:Hemolytic-Uremic Syndrome / Q000378:metabolism / Q000382:microbiology; D007684:Kidney Tubules / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D016612:Paraffin Embedding / Q000379:methods*; D022603:Shiga Toxins / Q000378:metabolism; D054323:Shiga-Toxigenic Escherichia coli / Q000378:metabolism; D014281:Trihexosylceramides / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D022603:Shiga Toxins; D014281:Trihexosylceramides; C018549:globotriaosylceramide