Confocal imaging of microglial cell dynamics in hippocampal slice cultures

Methods. 1999 Jun;18(2):222-30, 177. doi: 10.1006/meth.1999.0775.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Dissection / methods
  • Endocytosis
  • Fluorescein-5-isothiocyanate
  • Fluorescent Dyes
  • Hippocampus / cytology*
  • Hippocampus / physiology*
  • Lectins
  • Microglia / cytology*
  • Microglia / physiology*
  • Microscopy, Confocal / methods
  • Neuroglia / cytology
  • Neurons / cytology
  • Organ Culture Techniques / instrumentation
  • Organ Culture Techniques / methods
  • Rats
  • Rodentia

Substances

  • Fluorescent Dyes
  • Lectins
  • Fluorescein-5-isothiocyanate