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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Imaging the Gut with "CLARITY"

Published: June 11th, 2021

DOI:

10.3791/62143

1Department of Pathology & Laboratory Medicine, Emory University, Atlanta, GA

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 °C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 °C. On day 3, the tissue-hydrogel solution is incubated at 37 °C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 °C for 2 days (days 4 & 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4°C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide (hydrogel) embedding and tissue delipidation to preserve the 3-D tissue structure for immunostaining (without sectioning)1,2. The hydrogel-embedded tissue is optically transparent and macromolecule-permeable, with proteins and nucleic acids being preserved after removal of lipids by detergent. CLARITY was recognized as one among ten notable breakthroughs in 2013 by Science3Although developed initially to....

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All animal experiments described were approved by the Emory University Committee on the Use and Care of Animals. C57BL/6 mice (both male and female can be used) at 8-12 weeks of age were allowed free access to the food and water prior to euthanasia.

1. Removal of intestine (Day 1)

  1. Euthanize the mouse by carbon dioxide asphyxiation method, at a flowrate of 1.6 mL of carbon dioxide gas until cessation of breathing is observed for 2 min).
  2. Place the mouse supine on a dissecti.......

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The images from the CLARITY-cleared mouse gut tissue are represented in Figure 1. A successful completion of the protocol yields high quality, crisp images where all cellular details can be visualized clearly. The DAPI staining for nuclei is a very good index to assess the quality of the CLARITY protocol and the subsequent immunostaining as it can depict the tissue integrity. Further, the cell shape also provides a clue as to how the protocol has been successful especially in case of neurona.......

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The CLARITY method is highly useful for staining mouse gut to visualize various cell types including epithelia, neurons, and glial cells in 3D, especially the network of neuronal projections that extend across the gut wall to the lumen5 and their innervation to glial and EEC cells. The method presented here was modified according to the original study by Yang et al. 20141.

The critical steps in the protocol include immediate rinsing off the fixat.......

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The authors wish to acknowledge support from American Gastroenterology Association (AGA) AGA-Rome Functional GI motility Disorders Pilot Research Award (to BC), U.S. National Institutes of Health grant AI64462 (ASN) and the Emory University Integrated Cellular Imaging Microscopy Core.

....

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Name Company Catalog Number Comments
4% paraformaldehyde (PFA)  in PBS ThermoFischer Scientific J19943 Used for tissue fixation
4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) ThermoFischer Scientific D1306 1/10,000 dilution
Alexa Fluor-488 ThermoFischer Scientific A-11034 1/1000 dilution
Alexa Fluor-555 ThermoFischer Scientific A-21428 1/500 dilution
Anti- GFAP (Glial Fibrillary Acid Protein) Abcam ab7260 1/500 dilution
Anti-beta III Tubulin (or Tuj1) Abcam ab18207 1/1000 dilution
Chromogranin A Abcam ab15160 1/250 dilution
ProtoGel  30% National Diagnostics EC-890 Toxic, hazardous, handle with care- make 4% solution in PBS for use
Sodium dodecyl sulfate ThermoFischer Scientific 28364 8% in PBS
Vectashield mounting medium Vector Laboratories H-1000

  1. Yang, B., et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell. 158, 945-958 (2014).
  2. Chung, K., et al. Structural and molecular interrogation of intact biological systems. Nature. 497, 332-337 (2013).
  3. 2013 Runners-Up. CLARITY makes it perfectly clear. Science. 342, 1434-1435 (2013).
  4. Fung, T. C. The microbiota-immune axis as a central mediator of gut-brain communication. Neurobiology of Disease. 136, 104714 (2020).
  5. Chandrasekharan, B., et al. Interactions Between Commensal Bacteria and Enteric Neurons, via FPR1 Induction of ROS, Increase Gastrointestinal Motility in Mice. Gastroenterology. , (2019).
  6. Sylwestrak, E. L., Rajasethupathy, P., Wright, M. A., Jaffe, A., Deisseroth, K. Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution. Cell. 164, 792-804 (2016).
  7. Muntifering, M., et al. Clearing for Deep Tissue Imaging. Current Protocols in Cytometry. 86, 38 (2018).
  8. Cronan, M. R., et al. CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections. Disease Models & Mechanisms. 8, 1643-1650 (2015).
  9. Erturk, A., et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature Protocols. 7, 1983-1995 (2012).
  10. Qi, Y., et al. FDISCO: Advanced solvent-based clearing method for imaging whole organs. Science Advances. 5, 8355 (2019).
  11. Milgroom, A., Ralston, E. Clearing skeletal muscle with CLARITY for light microscopy imaging. Cell Biology International. 40, 478-483 (2016).
  12. Hu, W., Tamadon, A., Hsueh, A. J. W., Feng, Y. Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary. Journal of Visualized Experiments. , (2017).
  13. Chen, Y., et al. Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues. Scientific Reports. 9, 5624 (2019).

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