Identification and three-dimensional reconstruction of oxytocin receptor expressing astrocytes in the rat and mouse brain

Summary

Here, we present a step-by-step protocol for three-dimensional reconstruction of astrocyte morphology, applied to the central amygdala oxytocin receptor-expressing astrocytes. This includes RNAse-free perfusion, combination of RNAscope and immunohistochemistry, and confocal imaging. This protocol provides detailed information about tissue handling and a comprehensive description of the RNAScope technique to label rat and mouse oxytocin receptor mRNA. We also describe three-dimensional reconstruction that allows the assessment of more than 70 different cellular parameters, powerful for studying astrocyte morphology and astrocyte-astrocyte interactions.

For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021) and Althammer et al. (2020).

Graphical abstract

Highlights

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  RNAse-free perfusion and brain extraction from rat or mouse

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  Astrocyte-specific immunohistochemistry and confocal image acquisition

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  RNAScope in situ hybridization for oxytocin receptor mRNA

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  Three-dimensional reconstruction of oxytocin-receptor expressing astrocytes

Abstract

Here we present a step-by-step protocol for three-dimensional reconstruction of astrocyte morphology, applied to the central amygdala oxytocin receptor-expressing astrocytes. This includes RNAse-free perfusion, combination of RNAscope and immunohistochemistry, and confocal imaging. This protocol provides detailed information about tissue handling and a comprehensive description of the RNAScope technique to label rat and mouse oxytocin receptor mRNA. We also describe three-dimensional reconstruction that allows the assessment of more than 70 different cellular parameters, powerful for studying astrocyte morphology and astrocyte-astrocyte interactions.