Confocal imaging of microglial cell dynamics in hippocampal slice cultures

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.