title: A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.
abstract: This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Wiley
authors.affiliation: Departments of Ophthalmology & Visual Sciences and Cell Biology and Physiology, Washington University School of Medicine, Washington University, St. Louis, Missouri.
authors.forename: David C
authors.initials: DC
authors.lastname: Beebe
authors.affiliation: Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Mullins
authors.affiliation: Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Stone
authors.affiliation: Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Budd A
authors.initials: BA
authors.lastname: Tucker
mesh_terms: D000906:Antibodies / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry / Q000379:methods*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D018613:Microscopy, Confocal; D008867:Microtomy / Q000379:methods*; D009940:Organoids / Q000166:cytology*; D016612:Paraffin Embedding; D013194:Staining and Labeling
chemical_list: D000906:Antibodies
keywords: agarose; stem cell-derived 3-D organoids; thick sections