Identification of phagocytic glial cells after lesion-induced anterograde degeneration using double-fluorescence labeling: combination of axonal tracing and lectin or immunostaining

摘要: Retrograde and anterograde degeneration have been reported to be sufficient stimuli activate glial cells, which, in turn, are involved phagocytosis of degenerating material. Here we describe a double-fluorescence technique which allows for direct simultaneous visualization both labeled incorporated axonal debris incorporating cells the course degeneration. Stereotaxic application small crystals biotinylated tetramethylrhodamine (TRITC)-conjugated dextran amine Mini Ruby into medial entorhinal cortex resulted stable rhodamine fluorescence confined fibers terminals middle molecular layer dentate gyrus, stratum lacunosum-moleculare, crossed temporo-hippocampal pathway. Subsequent stereotaxic lesion induced transformation rhodamine-fluorescent granules. Incorporation these granules by microglial [labeled fluorescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolectin B4] or astrocytes (labeled FITC-coupled fibrillary acidic protein antibodies) phagocytosis-dependent labeling non-neuronal could identified microscopy. Electron microscopical analysis revealed that, following lesion, tracer remained axons were found cells. Our data show that TRITC- biotin-conjugated amines versatile tracers leading Phaseolus vulgaris leucoagglutinin-like staining. Lesion-induced anterogradely immunocytochemically can directly observed this on light electron levels.