Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Summary

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Abstract

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images of an entire adipose tissue.

Introduction

Until recently, adipose tissue was conceived of as an amorphous collection of fat cells. Over the past few decades, our understanding has grown more sophisticated, with fat now recognized to be a complex organ containing different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, the vasculature, and nerve projections. Interactions among these adipose-resident cells have pronounced effects on adipose tissue and organismal physiology and pathophysiology¹. Although emerging studies have unraveled important molecular mechanisms underlying certain interactions, a more comprehensive understanding requires reliable structural profiling of the entire tissue in three dimensions (3D).

Our current knowledge of adipose tissue morphology is largely based on histological analysis of thin sections (5 μm) with relatively high-magnification imaging (more than 10X)^2,3. However, this approach has several significant limitations. First, intricate filamentous structures such as sympathetic nerves and the vasculature, which are known to play important roles in adipose function^4,5,6,7, are difficult to evaluate through thin sections. Second, due to its seemingly amorphous shape and the lack of representative structural units to focus on, it is difficult to appreciate adipose tissue structures based only on section staining. Third, adipose tissue has a very high lipid content, creating challenges in obtaining consistent serial sections that are suitable for 3D anatomical reconstruction, a conventional method used to study whole brain morphology⁸. Given these factors, there is a great need for a whole-mount approach that can provide 3D visualization of an entire adipose depot while still achieving cellular resolution.

3D volumetric imaging of an entire organ is challenging due to the obscuring effects of light scatter. A major source of light scatter in biological tissues comes from lipid-aqueous interfaces. Although the efforts to eliminate scatter by removing lipids have been ongoing for over a century, there have been a large number of recent innovations⁹. One such newly developed tissue-clearing method is immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO/iDISCO+)^10,11. However, adipose tissue presents a particular challenge given its high level of lipids, and therefore, additional modifications to the iDISCO/iDISCO+ protocol are required to fully extract the lipids while protecting the tissue from collapsing. The modified protocol we have developed, now called Adipo-Clear, employs methanol/dichloromethane-based delipidation of adipose tissue to achieve optimal transparency suitable for high-resolution volumetric imaging¹². Because the delipidation step largely quenches endogenously expressed fluorescent proteins such as GFP and RFP, visualization of such proteins must be achieved by immunolabeling. Overall, this simple and robust protocol can be applied to study tissue-level organization of adipose-resident cells, lineage tracing of adipocyte progenitor cells, and adipose morphogenesis during development.

Protocol

Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee at the Rockefeller University.

1. Tissue Preparation

1. Perform standard intracardiac perfusion with ~20 mL of 1x phosphate buffered saline (PBS) at 4 °C until the blood is completely removed from the tissue.
2. Switch the perfusate to ~20 mL of fixative solution (4% paraformaldehyde (PFA) in 1x PBS) at 4 °C until the neck and tail have significantly stiffened.
   CAUTION: PFA is toxic. Avoid contact with skin, eyes, and mucous membrane. Solutions should be made inside a fume hood.
3. Dissect the fat pads of interest carefully to remove the entire fat pad without damaging it. Use blunt-ended forceps to avoid pinching or squeezing the tissue. Avoid contaminating the dissected tissue with any fur.
4. Post-fix the tissue in 4% PFA in 1x PBS overnight at 4 °C. For each fat pad, post-fix with ~10 mL of fixation solution in a 15 mL conical tube.
5. Wash the tissue 3 times (1 h each) with 1x PBS at room temperature (RT).
6. Store the tissue for short-term (up to 2 weeks) in 1x PBS at 4 °C and protected from light. For long-term storage (e.g., 1-2 years), switch the buffer to 1x PBS with 0.05% sodium azide (as a preservative).
   CAUTION: Sodium azide is highly toxic when ingested orally or absorbed through the skin. Concentrated sodium azide solutions (at 5% or greater) should be handled under a fume hood.

2. Delipidation and Permeabilization

Timing: 1-2 days

1. Prepare 20%, 40%, 60%, and 80% methanol gradient with B1n buffer (Table 1) (v/v), e.g., for 20% methanol/B1n buffer, mix 20 mL of methanol and 80 mL of B1n buffer. Store all buffers at 4 °C. Glycine crystals will precipitate from 60% and 80% methanol/B1n buffers due to saturation. Avoid removing the crystals; use the liquid solution for the following washes.
   CAUTION: Methanol is volatile, irritant, and flammable. Avoid skin or eye contact.
2. Gently remove the hair from the sample under a dissection microscope. Any hair, lint, or debris attached to the sample will cause shadows during imaging. Transfer the cleaned sample into a new tube.
3. Perform all following steps on ice or at 4 °C unless otherwise indicated. For small samples (e.g., posterior subcutaneous/perigonadal fat pads from young lean mice), use 2 mL microcentrifuge tubes with 1.6 mL of the solution. For large samples or samples with high lipid content (e.g., fat pads from obese mice), use 5 mL tubes with 4 mL of the solution.
   1. Place the tubes containing the samples horizontally on an orbital shaker set at ~ 100 rpm. Make sure the samples can move freely inside the tube.
4. Dehydrate the sample in a graded series of 20%, 40%, 60%, 80%, and 100% methanol/B1n buffer for the indicated time (Table 2). Minimize the carry-over solution.
5. Delipidate the sample with 100% dichloromethane (DCM) (3 times), each with the incubation time indicated in Table 2. The sample should sink to the bottom of the tube at the end of the second and third DCM washes. If not, extend the incubation time to ensure full delipidation (e.g., extend from 30 min to 1-2 h).
   CAUTION: DCM should be handled inside a fume hood. Use glass containers for storage and microcentrifuge tubes that are made from polypropylene for incubations. The microcentrifuge tubes should not be used for long-term storage of DCM. Petri dishes and serological pipettes that are made from polystyrene are not compatible with DCM. DCM is highly volatile. Transfer the sample into fresh solution quickly to prevent desiccation.
6. Wash twice with 100% methanol for the indicated time (Table 2).
7. Optional: If the sample is not well-perfused, the color of hemoglobin from the remaining red blood cells might cause strong autofluorescence. Bleach with 5% H₂O₂ in methanol (1 volume of 30% H₂O₂ to 5 volumes of methanol) overnight at 4 °C.
8. Rehydrate the sample in a reversed methanol/B1n buffer series: 80%, 60%, 40%, 20% methanol/B1n, and 100% B1n buffer (2 times) for the indicated time (Table 2).
9. Wash the sample with PTxwH buffer (Table 1) for 2 h at RT.
10. Store the delipidated sample in PTxwH buffer at 4° C (up to 1 year) or proceed immediately to the immunostaining step.

3. Whole Mount Immunostaining

Timing: 8-10 days

Note: All of the following steps should be carried out at RT unless noted otherwise, with shaking and protection from light. For small samples, use 2 mL microcentrifuge tubes with 1.6 mL of solution. For large samples, use 5 mL tubes with 4 mL of solution. It is recommended to first validate the antibodies on small pieces of the tissue or methanol-treated tissue sections.

1. Dilute the primary antibody in PTxwH buffer to the recommended concentration. Centrifuge the diluted antibody solution at ~20,000 x g for 10 min to prevent introducing antibody precipitates or aggregations.
2. Incubate the delipidated sample with the primary antibody solution for the indicated time (Table 2).
3. Wash the sample with PTxwH buffer in a series of incubation steps: 5 min, 10 min, 15 min, 20 min, 1 h, 2 h, 4 h, and overnight.
4. Dilute the secondary antibody in PTxwH buffer to the recommended concentration. Centrifuge the diluted antibody solution at ~20,000 x g for 10 min to prevent introducing antibody precipitates or aggregations.
   Note: To avoid high background caused by tissue autofluorescence, secondary antibodies conjugated with fluorophores that emit light in the red and far-red regions are recommended. Depending on the number of laser lines available on a microscope, up to 3-4 markers can be imaged simultaneously in one sample.
5. Incubate the sample with the secondary antibody solution for the indicated time (Table 2).
6. Wash sample with PTxwH buffer in a series of incubation steps: 5 min, 10 min, 15 min, 20 min, 1 h, 2 h, 4 h, and overnight.
7. Optional: For tissue types that are fragile, fix the stained tissue with 4% PFA in 1x PBS overnight at 4 °C to help preserve tissue morphology.
   Note: This step may increase imaging background. It is not necessary to perform this step for adipose tissue.
8. Wash the sample with 1x PBS in a series of incubation steps: 5 min, 10 min, and 30 min.
9. Gently remove lint from the sample under a dissection microscope.

4. Tissue Clearing

Timing: 1-2 days

1. Optional: Embed the sample in agarose to facilitate sample mounting for light-sheet microscopy.
   Note: This step is strongly recommended for adipose tissue to stabilize its shape during imaging.
   1. Prepare the embedding solution with 1% agarose in 1x PBS (w/v). Cool the embedding solution to ~ 40 °C to avoid exposing the sample to excessive heat.
   2. Place the cleaned sample into a mold (e.g., Petri dish or weighing boat) and arrange it into the desired position. Avoid any liquid carryover. Gently pour the embedding solution over the sample. Avoid any air bubbles.
   3. Let the agarose fully solidify at RT. Cut out a block containing the sample.
      Note: All of the following steps including overnight incubations should be carried out at RT, with shaking and protection from light. For small samples, use 2 mL microcentrifuge tubes with 1.6 mL of solution. For large samples, use 5 mL tubes with 4 mL of solution.
2. Dehydrate the sample in methanol gradient with H₂O: 25%, 50%, 75%, and 100% (3 times) for the indicated time (Table 2).
   Note: Sample can be left overnight at the 100% methanol step.
3. Incubate the sample in 100% DCM for 1 h with shaking (3 times). Sample should sink to the bottom of the tube at the end of each DCM wash. If not, extend the incubation time to ensure full removal of methanol. Sample can be left overnight at the 100% DCM step.
4. Incubate the sample in dibenzyl ether (DBE) overnight with mild shaking to achieve refractive index matching.
   Note: Sample will eventually become transparent in visible light.
   CAUTION: DBE is hazardous. Avoid any contact with skin. Handle it inside a fume hood with double-layered gloves. Discard the gloves as soon as it is contaminated with DBE. Use glass containers for storage and microcentrifuge tubes (polypropylene) for incubations. The microcentrifuge tubes should not be used for long-term storage of DBE. Petri dishes and serological pipettes that are made from polystyrene are not compatible with DBE. Clean any spill with 100% ethanol.
5. Incubate the sample with fresh DBE with mild shaking for 2 h. Store the sample at RT in the dark or proceed directly to imaging.
   Note: Samples are recommended to be imaged within a month. Although certain fluorophores are more stable than others, long-term storage of the samples at this stage is not recommended.

5. Microscopy

1. Imaging with a light-sheet microscope that is compatible with DBE.
   Note: Only objectives that are approved for organic solvent-based imaging and matched with the refractive index of DBE can be used as dipping objectives in DBE-immersed imaging.
   1. Mount the agarose-embedded sample onto a holder that can prevent movement during imaging. Immerse the sample into a DBE-filled chamber.
   2. Collect a z-stack covering the whole sample (e.g., 1-2X magnification) to obtain whole-tissue distribution/structure of the marker of interest.
   3. Switch to an objective with higher magnification (e.g., 4-12X magnification) to zoom in on the regions of interest to image detailed structures.
      Note: Collagen is a major contributor to the extracellular matrix of adipose tissue, which surrounds all fat cells¹³. Collagen has a typical emission spectrum ranging around 400 nm to 550 nm¹⁴. Imaging the sample with the 488 laser line (green) and collecting the emitted light at a wavelength range that lies within the emission spectrum of collagen (e.g., 525-550 nm) will provide the tissue autofluorescence signal, which was previously shown to delineate fat cell contour and overall tissue architecture¹².
2. Imaging with an inverted confocal or a two-photon microscope.
   1. Place the agarose-embedded sample into a chamber slide with a glass bottom without touching any plastic parts (or other DBE-compatible imaging chamber that can seal). Make sure DBE does not leak out.
   2. Choose the objectives with longer working distances to achieve deeper penetration.
      Note: Imaging should be done with an inverted confocal or two-photon microscope through the glass bottom of the chamber slide. Avoid direct contact between the sample and dipping objectives that are not suggested for DBE-based imaging.

Results

Adipo-Clear prepared whole fat pads can be imaged in 3D to analyze how tissue morphology and cellular interactions are affected in the lean and obese states. This method can be easily applied to analyze general adipose structure by collecting the tissue autofluorescence signal in the green channel. We have previously shown that the autofluorescence signal in adipose overlays favorably with perilipin staining, a commonly used marker to outline mature adipocytes¹². F...

Discussion

Adipo-Clear is a straightforward and robust method for clearing adipose tissue, which can be easily performed in a regular lab setup. In comparison to other solvent-based clearing methods such as iDISCO/iDISCO+^10,11,12, Adipo-Clear is particularly optimized for clearing adipose tissue and other tissue with high fat content. The delipidation step completely removes lipids from adipose, and therefore facilitates immunolabeling thr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x phosphate buffered saline	Corning	21-040-CV
Paraformaldehyde	Sigma Aldrich	P6148-1KG
Methanol	Fisher Scientific	A412SK-4
Triton X-100	Sigma Aldrich	X100-500ML
Tween 20	Sigma Aldrich	P2287-500ML
Heparin	Sigma Aldrich	H3393-100KU
Dichloromethane	Sigma Aldrich	270991
Hydrogen peroxide 30%	Fisher Scientific	325-100
Benzyl ether	Sigma Aldrich	108014
Agarose	Invitrogen	16500500
Sodium azide	Sigma Aldrich	71289-5G
Glycine	Fisher Scientific	BP381-1
Rabbit polyclonal anti-Tyrosine Hydroxylase	Millipore	AB152	1:200 dilution
Goat polyclonal anti-CD31/PECAM-1	R&D Systems	AF3628	Final concentration of 2 µg/mL
Rat monoclonal anti-CD68, Clone FA-11	Bio-Rad	MCA1957	Final concentration of 2 µg/mL
Donkey anti-rabbit IgG (H+L) Alexa Fluor 647	Jackson ImmunoResearch	711-605-152	Final concentration of 5-10 µg/mL
Donkey anti-goat IgG (H+L) Alexa Fluor 568	Invitrogen	A11077	Final concentration of 5-10 µg/mL
Donkey anti-rat IgG (H+L) Alexa Fluor 647	Jackson ImmunoResearch	712-605-153	Final concentration of 5-10 µg/mL
Imaging chamber	ibidi	80287
Light sheet microscope	LaVision BioTec	Ultramicroscope II
Imaging software	LaVision BioTec	Imspector software
Microscopy visualization software	Bitplane	Imaris