Two-Photon Microscopy of Astrocytes and Excitatory Neurons in a Cleared Mouse Hippocampal Tissue

Abstract

Source: Refaeli, R., et al., Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains. J. Vis. Exp. (2022)

The video demonstrates the preparation of a chamber and the neuroimaging of cleared mouse hippocampal tissue using two-photon microscopy to visualize the spatial interactions between fluorescently labeled astrocytes and excitatory neurons.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AAV1-GFAP::TdTomato	ELSC Vector Core Facility (EVCF)		Viral vector used to detect astrocytes
AAV5-CaMKII::eGFP	ELSC Vector Core Facility (EVCF)		Viral vector used to detect neurons
AAV5-CaMKII::H2B-eGFP	ELSC Vector Core Facility (EVCF)		Viral vector used to detect neuronal nuclei
AAV5-CaMKII::TdTomato	ELSC Vector Core Facility (EVCF)		Viral vector used to detect neurons
Imaris software	Bitplane, UK		A software that allows 3D analysis of images
RapiClear	SunJin lab	#RC147002
RapiClear CS	SunJin lab	#RCCS002
SyGlass software			A software that allows 3D analysis of images using virtual reality
Two photon microscope	Neurolabware		Ti:sapphire laser (Chameleon Discovery TPC, Coherent), GaAsP photo-multiplier tubes (Hamamatsu, H10770-40) , bandpass filter (Semrock), water immersion 16x objective (Nikon, 0.8 NA)
VA-044 Initiator	Wako	#011-19365