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

Gabriela De la Cruz; Nana Nikolaishvili Feinberg; Scott E Williams

doi:10.1007/978-1-0716-2752-5_7

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

Methods Mol Biol. 2023:2583:63-79. doi: 10.1007/978-1-0716-2752-5_7.

Authors

Gabriela De la Cruz^{1

2

3}, Nana Nikolaishvili Feinberg^{1

2

3}, Scott E Williams^{4

5

6}

Affiliations

¹ Department of Pathology & Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
² Pathology Services Core, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
³ Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁴ Department of Pathology & Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. scott_williams@med.unc.edu.
⁵ Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. scott_williams@med.unc.edu.
⁶ Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. scott_williams@med.unc.edu.

PMID: 36418726
DOI: 10.1007/978-1-0716-2752-5_7

Abstract

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.

Keywords: Asymmetric cell division; Cerebellar granule neuron progenitors; Cerebellum; Division orientation; Granule cells; Microcephaly; Mitosis; Oriented cell division; Spindle orientation.

MeSH terms

Animals
Brain
Fluorescent Antibody Technique
Mice
Neurogenesis* / physiology
Spindle Apparatus*
Staining and Labeling