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Cold Spring Harb Protoc. 2012 May 1;2012(5). pii: pdb.prot069286. doi: 10.1101/pdb.prot069286.

Optical window preparation for two-photon imaging of microglia in mice.


Microglia are the primary immune effector cells of the brain parenchyma. They are distributed throughout the brain at various densities. Two-photon fluorescence microscopy, together with expression of fluorescent proteins in microglia, has enabled the study of these fascinating cells in vivo. Imaging studies have shown, for example, that microglia continually survey their cellular environment and immediately respond to injury. However, we still know very little about their roles in various parts of the developing and adult brain or their diverse effector functions in aging and different disease states. Experimental procedures have been developed for minimally invasive short- and long-term two-photon imaging of microglial cells in cortical regions of the intact mouse brain. This protocol presents two methods for the preparation of the optical window that is needed for two-photon imaging of microglia. The thinned skull method should be used whenever possible. Skull thinning enables transcranial two-photon imaging while minimizing external influences that might disturb normal microglia physiology and brain homeostasis. The sealed craniotomy preparation is useful for short-term investigation of microglia.

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