Automated Immunofluorescence Staining for Analysis of Mitotic Stages and Division Orientation in Brain Sections

Microcephaly often results from mitotic defects in neuronal progenitors, frequently by decreasing proliferation rates or shifting cell fates. During neurogenesis, oriented cell division-the molecular control of mitotic spindle positioning to control the axis of division-represents an important mechanism to balance expansion of the progenitor pool with generating cellular diversity. While mostly studied in the context of cortical development, more recently, spindle orientation has emerged as a key player in the formation of other brain regions such as the cerebellum. Here we describe methods to perform automated dual-color fluorescent immunohistochemistry on murine cerebellar sections using the mitotic markers phospho-Histone H3 and Survivin, and detail analytical and statistical approaches to display and compare division orientation datasets.