This article discusses the preparation and staining of cortical tissue to visualize lipofuscin granules in neurons using transmission electron microscopy. Lipofuscin accumulation is associated with aging and various neuropathological conditions.
Begin with prefixed cortical tissue, where neurons contain lipofuscin granules. These lipid-rich granules accumulate with aging and are associated with neuropathological conditions.
Treat the tissue with osmium tetroxide to stabilize the cell structures, including lipofuscin.
Treat the tissue with increasing concentrations of ethanol to dehydrate it. Then, embed it in resin, and cut ultrathin sections to reveal neuronal structures.
Mount the section onto an uncoated copper grid.
Stain the section with uranyl acetate, a heavy-metal stain.
The uranyl ions interact with cellular biomolecules, including lipids, proteins, and DNA.
Next, stain the tissue with lead citrate, which interacts weakly with pre-bound uranyl ions, enhancing the contrast against the background.
Place the stained slice under a transmission electron microscope to expose it to a focused electron beam.
Stained biomolecules enriched with heavy-metal stains, scatter electrons strongly compared to the background.
Lipofuscin granules appear as irregular, dense clusters against the lighter cytoplasmic background, confirming their presence in the neurons.
繁體中文
