Large-scale ISH on mouse brain sections for systematic gene expression analysis in developing mouse diencephalon

The mammalian diencephalon gives rise to the thalamus and hypothalamus. The thalamus contains multiple sensory nuclei and relays sensory information to corresponding cortical areas, while the hypothalamus is a central regulator of many homeostatic behaviors that are essential for survival, such as temperature regulation, food intake, and circadian rhythms. However, despite the functional importance of these brain regions, the molecular mechanisms that guide specification and differentiation of neurons in specific thalamic and hypothalamic regions are still largely unknown. It is therefore crucial to obtain molecular markers for each nucleus and for individual subtypes of neural progenitors and precursors in these structures to obtain a better understanding of the development, connectivity, and function of diencephalic neurons. Here we will describe a protocol that we have previously used to conduct large-scale, high-quality twocolor ISH to characterize gene expression patterns in the developing mouse diencephalon.