Abstract
Methods are described for imaging the cellular dynamics of microglia in live mammalian brain slice cultures. Brain slices prepared from developing rat hippocampus are cultured for up to 2 weeks by the roller tube or static filter culture technique, stained with one or more fluorescent dyes, and imaged by scanning laser confocal microscopy. One of several cell type-specific or nonspecific fluorescent dyes can be used independently or in combination to label cells in live brain tissues. The fluorescently conjugated plant isolectin GSA-IB4 is useful for identifying microglia and for following their structure, movement, and proliferation. Live and dead neurons and glia can be distinguished using membrane-permeant and -impermeant fluorescent nucleic acid dyes. Nonspecific fluorescent lipids such as DiIC18 can be used as a vital stain to label populations of endocytic and phagocytic cells. Using multichannel confocal imaging, tissue slices that are single-, double-, or triple-labeled can be imaged in the living state in two or three spatial dimensions as well as in time. This provides a means for investigating the cell–cell interaction and dynamic behavior of microglia and other cell types in live brain tissues cultured under various physiological conditions.
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