Open AccessA Method for Sectioning and Immunohistochemical Analysis of Stem Cell–Derived 3‐D Organoids
Author(s) -
Wiley Luke A.,
Beebe David C.,
Mullins Robert F.,
Stone Edwin M.,
Tucker Budd A.
Publication year - 2016
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/cpsc.3
Subject(s) - vibratome , organoid , induced pluripotent stem cell , biology , microbiology and biotechnology , stem cell , immunohistochemistry , cell type , cell , agarose , biomedical engineering , anatomy , immunology , embryonic stem cell , biochemistry , medicine , gene
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.