Simple and Rapid Tissue Clearing Method for Three‐Dimensional Histology of the Pancreas

Hang Sheung Wong1, Patrick Ka Kit Yeung1, Hei Ming Lai1, Karen Siu Ling Lam2, Wu Wutian3, Sookja Kim Chung4

1 School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, 2 Department of Medicine, The University of Hong Kong, Hong Kong, 3 Joint Laboratory of Jinan University and The University of Hong Kong, GHM Institute of CNS Regeneration, Jinan University, Guangzhou, 4 Corresponding author (skchung@hku.hk)
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.20
DOI:  10.1002/cpcb.34
Online Posting Date:  December, 2017
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Abstract

Previously, high‐resolution three‐dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide‐based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel‐embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time‐consuming and less error‐prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild‐type and gene‐knockout 6‐week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP‐GFP). This technique could facilitate high‐resolution, three‐dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

Keywords: tissue clearing; CLARITY; simple; pancreas; three‐dimensional imaging

     
 
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Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:  

  Materials
  • MIP‐GFP mice (B6.Cg‐Tg(Ins‐EGFP) 1Hara/J, stock no. 006864)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 4% paraformaldehyde fixative (PFA) in PBS (see recipe)
  • 8% sodium dodecyl sulfate (SDS) in PBS (see recipe)
  • PBST (see recipe)
  • Primary and secondary immunostaining antibodies
  • Sodium azide or thiomersal (optional)
  • 88% (w/v) iohexol (see recipe)
  • 27‐G needle and 1‐ml syringe
  • Surgical instruments (e.g., blunt scissors, iris scissors, blunt forceps, hemostats)
  • 60‐ml syringe and perfusion set with blunt tipped 15‐G butterfly needle
  • Glass bottom microwell dishes; 35‐mm Petri dish, 20‐mm microwell (MatTek Corp.)
  • Additional reagents and equipment for injection (Donovan & Brown, ) and anesthesia (Donovan & Brown, ) of mice
NOTE: Animal handling should follow local institutional guidelines, and local practice may vary. In this protocol, use of MIP‐GFP mice was approved and handled in accordance with the guidelines provided by the Committee on the Use of Live Animals in Teaching and Research (CULATR) in the Laboratory Animal Unit, University of Hong Kong, with prior approval (CULATR reference number: 3175‐13).
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Figures

Videos

Literature Cited

Literature Cited
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  Chung, K., & Deisseroth, K. (2013). CLARITY for mapping the nervous system. Nature Methods, 10(6), 508–513. doi: 10.1038/nmeth.2481.
  Donovan, J., & Brown, P. (2001). Anesthesia. Current Protocols in Immunology, 27, 1.4.1–1.4.5. doi: 10.1002/0471142735.im0104s27.
  Donovan, J., & Brown, P. (2006). Parenteral injections. Current Protocols in Immunology, 73, 1.6.1–1.6.10. doi: 10.1002/0471142735.im0106s73.
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