A Method for Sectioning and Immunohistochemical Analysis of Stem Cell–Derived 3‐D Organoids

Luke A. Wiley1, David C. Beebe2, Robert F. Mullins1, Edwin M. Stone1, Budd A. Tucker1

1 Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 2 Departments of Ophthalmology & Visual Sciences and Cell Biology and Physiology, Washington University School of Medicine, Washington University, St. Louis, Missouri
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.19
DOI:  10.1002/cpsc.3
Online Posting Date:  May, 2016
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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.

Keywords: agarose; stem cell–derived 3‐D organoids; thick sections

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Embedding Stem Cell–Derived 3‐D Organoids In Low‐Melt Agarose
  • Basic Protocol 2: Sectioning Embedded Stem cell–Derived Organoids Using a Vibratome
  • Basic Protocol 3: Antibody Labeling and Staining of Stem cell–Derived Organoids Using Netwells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Embedding Stem Cell–Derived 3‐D Organoids In Low‐Melt Agarose

  Materials
  • Low‐melting‐temperature agarose (Research Products International Corp., cat. no. A20070‐100.0)
  • Phosphate buffered saline (PBS; Thermo Fisher Scientific, cat. no. 10010‐023)
  • Stem cell–derived 3‐D organoids (Eiraku et al., ; Spence et al., ; Nakano et al., ; Lancaster et al., ; Zhong et al., ; Beauchamp et al., ; Dye et al., ; Small et al., )
  • 500‐ml glass beaker
  • LabDoctor Hotplate Magnetic Stirrer (Midwest Scientific, cat. no. SH‐1500;) or equivalent
  • Large stir bar
  • Microwave oven
  • 50‐ml polypropylene conical tubes (Sarstedt, cat. no. 62.559.010)
  • 50°C water bath
  • 35 × 10–mm Falcon disposable petri dishes (Corning Life Sciences, cat. no. 25373‐041)
  • Small laboratory tissues (e.g., Kimwipes)
  • Metal forceps (e.g., Dumont #5 forceps; Fine Science Tools, cat. no. 11251‐10)

Basic Protocol 2: Sectioning Embedded Stem cell–Derived Organoids Using a Vibratome

  Materials
  • 3‐D organoids embedded in 4% agarose (from protocol 1)
  • Super glue
  • Sectioning buffer (see recipe)
  • Metal spatula (e.g., Sigma‐Aldrich, cat. no. Z283274)
  • Razor blades (Stanley, cat. no. 11‐515)
  • Double‐edged carbon steel Feather blades (Ted Pella, cat. no. 121‐15)
  • Two pairs of metal forceps (suggest Dumont #5 forceps; Fine Science Tools, cat. no. 11251‐10 and Dumont #55 forceps; Fine Science Tools, cat. no. 11295‐51)
  • Vibrating tissue slicer or vibratome (Leica VT1000 S Vibratome, Leica Microsystems) or equivalent
  • Dissecting light microscope (Carl Zeiss Stemi DV4 Series Stereomicroscope) or equivalent
  • Microscopy fiber optic illuminator (Cole Parmer, cat. no. EW‐41723‐30) or equivalent

Basic Protocol 3: Antibody Labeling and Staining of Stem cell–Derived Organoids Using Netwells

  Materials
  • Thick agarose sections of stem cell–derived 3‐D organoids ( protocol 2)
  • Primary antibodies of interest
  • Blocking buffer (see recipe)
  • Wash buffer: phosphate‐buffered saline (Thermo Fisher Scientific, cat. no. 10010‐023) containing 0.2% (v/v) Tween 20 (Sigma‐Aldrich, cat. no. P2287)
  • Species‐appropriate, fluorescently conjugated secondary antibody of interest
  • Mounting medium (see recipe)
  • 15‐mm Netwells inserts (Corning, cat. no. 3477)
  • Polystyrene beaker cups (VWR International, cat. no. 13915‐985)
  • Netwells 12‐well carrier kit for 15‐mm inserts (Corning, cat. no. 3520)
  • Netwells reagent trays, black (Corning, cat. no. 3517)
  • Dissecting light microscope (Carl Zeiss Stemi DV4 Series Stereomicroscope, Carl Zeiss) or equivalent
  • Microscopy fiber optic illuminator (Cole Parmer, cat. no. EW‐41723‐30) or equivalent
  • Metal forceps (e.g., Dumont #55 forceps; Fine Science Tools, cat. no. 11295‐51)
  • Metal spatula (e.g., Sigma‐Aldrich, cat. no. Z283274)
  • 25 × 75–mm glass coverslips (Nalge Nunc International, cat. no. 171080)
  • Leica DM 2500 SPE confocal microscope (Leica Microsystems) or similar
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Figures

Videos

Literature Cited

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