Imaging the Gut with "CLARITY"

Summary

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.

Abstract

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.

Introduction

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 Science³. Although developed initially to....

Protocol

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 dissecting board with limbs pinned. Sterilize the abdomen with 70% ethanol. Using forceps, cut open the skin with scissors.
3. Lift the liver gently an....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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