A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
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 Science3. Although developed initially to image lipid-rich brain tissues where lipids affect light scattering and imaging quality, the technique is currently widely used for imaging other tissues like intestine, liver, kidney and heart. The technique offers the ability to stain and image a tissue by eliminating the need for sectioning, which otherwise might result in a biased evaluation of cell-cell interactions due to irregular distribution of cell types. The technique also provides the opportunity to perform confocal z-stacks and recreate 3-dimensional image of the tissue, which can help determine the densities, microarchitecture, and cell-cell interaction among various cell types more realistically than from a tissue section. Moreover, the technique has been modified to allow immunostaining of dense tissues like bone, as well as in situ hybridization studies. The hydrogel embedded 3D tissue offers an attractive platform for elucidating the interactions between epithelial, enteroendocrine, glial and neuronal cells, which have been recently cited to be crucial to the understanding of pathophysiology of diseases like Parkinson's Disease, Alzheimer's, Autism Spectrum Disorders etc4.
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)
2. Fixation of intestinal tissue (Day 1)
3. Hydrogel embedding (Day 2)
4. Hydrogel polymerization and delipidation (clearing) step (Day 3)
5. Washing off the detergent from the cleared tissue (Day 6)
6. Immunofluorescent staining for neurons, glia and enteroendocrine cells
7. Confocal Imaging
NOTE: The cleared, stained and mounted tissue can be imaged immediately or can be stored in slide boxes at -20°C.
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...
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...
The authors have nothing to disclose.
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.
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 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved