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 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.

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 and identify the stomach. Then holding the stomach with forceps, cut off the esophagus just above the stomach. Holding the stomach with forceps, gently snip away the mesenteries and detach the intestine from the abdominal cavity up to the rectum. Cut off the detached intestine at the rectum.
4. Transfer the detached intestine into an ice-cold PBS solution in a Petri dish. Now gently snip off the mesentery between the ileal segments and straighten out the intestine so that starting from the stomach all the mesenteries are snipped off, up to the colon.
5. Cut 1-1.5 cm of the desired intestinal region for CLARITY staining into a new Petri dish containing ice-cold PBS.
6. Using a 5 mL syringe attached to a blunt end of a 20 G needle, flush out the fecal contents with ice cold PBS.
   ​NOTE: Any region of the gut can be used for CLARITY staining using the same technique. The intestinal segment may be cut open along the mesentery or used intact for the subsequent steps.

2. Fixation of intestinal tissue (Day 1)

1. Fix the tissue in 4% paraformaldehyde (PFA) solution in PBS.
2. Transfer 1-1.5 cm piece of intestinal segment of interest into a 15 mL conical tube filled with 4% PFA at 4 °C overnight for fixation.

3. Hydrogel embedding (Day 2)

1. Transfer the intestinal segment from 4% PFA solution with forceps into a new 15 mL conical tube with PBS solution and wash three times (5 min each, on a shaker at 150-200 rpm) to remove any residual PFA.
2. Preparation of 4% hydrogel
   1. Use the 30% gel solution (ProtoGel) to prepare the hydrogel. For preparing 4% hydrogel solution, dilute the 30% stock solution is diluted in PBS. To prepare 12 mL of 4% hydrogel, take 1.6 mL of 30% gel solution and add 10.4 mL of PBS.
3. Transfer the fixed tissue from step 3.1. to the 4% hydrogel solution in PBS in a 15 ml conical tube and incubated at 4 °C overnight.
   NOTE: It is highly recommended that the tissue is washed free of PFA using PBS after overnight incubation. Tissue stored longer in PFA does not give good results with subsequent steps and staining. The fixed tissue after PBS washes may be stored at 4 °C for a week.

4. Hydrogel polymerization and delipidation (clearing) step (Day 3)

1. Take the conical tube containing the tissue in hydrogel solution out from 4 °C. Transfer to a 37 °C water bath and incubate for 1 h. This step allows for hydrogel polymerization.
2. After 1 h, take out the conical tube containing the hydrogel embedded tissue of the water bath, and pour off the acrylamide solution. Now gently rinse the tissue with a single PBS wash at room temperature to remove the excess hydrogel.
3. Delipidation step
   1. Delipidate by incubating the tissue in sodium dodecyl sulfate (8% SDS) in PBS. Transfer the hydrogel embedded tissue from step 4.2 to an 8% SDS solution in a 50 mL conical tube. Make sure that the tissue is completely immersed in the SDS solution, and that the tube is capped.
   2. Transfer the capped 50 mL tube containing the tissue in SDS onto a 37 °C shaker (200 rpm) at room temperature for 2 days (Days 4 & 5) to allow delipidation.
      NOTE: Ensure that SDS solution is filled up to at least 20-25 mL in the 50 mL conical tube, to compensate for any evaporation that might occur at 37 °C during the 2 days of incubation.
4. On days 4 & 5, incubate the tissue in SDS at 37 °C shaker (200 rpm) at room temperature.

5. Washing off the detergent from the cleared tissue (Day 6)

1. Take the tissue from the SDS solution. The tissue will appear 'clear' or transparent.
2. Transfer the tissue to a new 50 mL comical tube with PBS solution and wash over the course of the day on a shaker (150-200 rpm) at room temperature with several changes of PBS, to ensure that all traces of SDS are removed. At least 10 changes of PBS solution will ensure that tissue is free off the SDS, and a good rule of thumb is to check if the final PBS wash has no traces of froth/ foam from the SDS. This is a critical step as SDS can interfere with the subsequent staining steps.
   NOTE: At the end of the day, the cleared tissue after several washes in PBS is now ready for immunostaining or maybe stored at 4 °C for up to 3 weeks.
3. Storing cleared intestinal tissues for more than a month in PBS solution will affect the tissue integrity and quality of staining. Use the cleared intestinal tissue immediately or within 1-2 weeks of clearing for best results.

6. Immunofluorescent staining for neurons, glia and enteroendocrine cells

1. Transfer the cleared tissue to a 1.5 or 2 mL tube and incubate in 500 µL volume of respective primary antibodies diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100) overnight at 4 °C. The antibodies (Ab) used: Tuj1 or β-3-tubulin (Pan neuronal marker, 1: 1000), Glial Fibrillary Acid Protein (GFAP, 1: 500), Chromogranin A (1: 250).
2. After overnight incubation, subject the tissue to 3 PBS washes (5 min each using 1-1.5 mL of PBS) to remove unbound primary Ab.
3. Transfer the tissue to a new tube, and subsequently incubate with 500 µL of appropriate secondary Alexa Fluor Ab (AF) for 1.5 h at room temperature in the dark. The secondary Ab used are AF 488 for Tuj1 (1: 1000), AF-555 for GFAP (1: 500) and AF 555 (1 :250) for Chromogranin A.
4. After incubation, wash the tissue thrice with PBS as in Step 6.2.
5. For staining the nuclei, incubate the tissue in DAPI (1: 10000 dilution or 0.2 µL in 2 mL PBS) for 5 min at room temperature.
6. Finally, transfer the stained tissue onto a clean glass slide, add coverslip on top of the tissue and mount using 100 µL of VectaShield for confocal imaging.
7. Place the slides in the dark at room temperature in a slide holder for 30 min for drying, after which they can be imaged or stored at -20 °C. Mouse gut wall is fairly thin and once opened along the mesentery, it can be mounted on glass slides directly with coverslip. If working with larger tissues like spleen, kidneys, chamber (cavity) slides or spacers may be used. For bigger tissues like, a cavity slide may be used for mounting.
   ​NOTE: It is to be noted that if the intestinal segment was not cut open at the beginning step (refer to Note in Step 1), it is advisable to cut open the intestinal tube with micro scissors that will facilitate the steps involving mounting.

7. Confocal Imaging

NOTE: The cleared, stained and mounted tissue can be imaged immediately or can be stored in slide boxes at -20°C.

1. Image using a confocal microscope (e.g., Olympus FV1000).
2. Take z-stacks across 50 µm thickness of the gut tissue to visualize the regions from the gut lumen toward the serosal layer.
3. Store the z stack images as image files, .avi movie files or convert into 3D image with the help of the software⁵.

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