Intracellular Lucifer Yellow staining and electron microscopy of neurones in slices of fixed epitumourous human cortical tissue

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Abstract
To examine the complete morphology and ultrastructure of lipofuscin-containing human pyramidal cells, epitumourous biopsy tissue was lightly fixed in paraformaldehyde. Cortical slice preparations were immersed in an injection chamber which was transferred to a fixed stage microscope. Electrodes were filled with an aqueous solution of the fluorescent dye Lucifer Yellow and attached to a micromanipulator. Epifluorescence illumination was used to visualize and guide the tip of the Lucifer pipette towards lipofuscin-containing, autofluorescent pyramidal cell somata. After impaling, the neuron was intracellularly stained by iontophoretic injection of Lucifer Yellow. Subsequent graphical reconstructions of injected pyramidal cells revealed complete filling of their dendritic arborizations. Comparison with published Golgi-material prepared for light microscopy revealed no patho-morphological changes in the tissue. Eventually, dye-filled cells were photo-oxidized in the presence of diaminobenzidine, which resulted in the formation of a homogeneously brown reaction product. Bleached cells were then osmicated and embedded in plastic. Electron microscopy revealed fine electron-opaque label distributed through-out the karyo- and cytoplasm and there were no apparent gross ultrastructural changes. Cytological details, such as organelles and membranes were not obscured by the reaction product. Due to its autofluorescence, the pigment part of the lipofuscin also underwent photoconversion, resulting in a highly enhanced electron-dense matrix. Due to its high selectivity and relative methodological simplicity, the approach presented is considered to be a promising alternative to the gold-toning modification of the Golgi-electron microscope technique.