A Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Abstract

Adipose tissue is not simply a collection of lipid storing fat cells, but is now recognized to be a complex tissue with a central role in whole body energy homeostasis. In order to understand how adipocytes and associated cell types interact in normal physiology and in pathological states like obesity, it is critical to obtain a holistic view of cells and structures in three dimensions. To that end, we have adapted the iDISCO/iDISCO+ tissue clearing protocol to facilitate the delipidation of fat tissues, while still maintaining their architecture. We describe here this method, that we refer to as Adipo-Clear, highlighting key steps in the protocol as well as important technical considerations. This versatile approach can provide entirely new insights into adipose tissue biology in health and disease.

1 Introduction

Adipose tissue was originally thought of as a simple collection of adipocytes or fat cells that serve as a passive repository for lipid storage. However, fat is now appreciated to be a complex tissue containing diverse cell types, including adipocytes, preadipocytes, immune cells, blood vessels, nerve projections, and fibroblasts. Together, these cells contribute to the remarkable capacity of adipose tissue to remodel in response to changes in nutritional status. Moreover, thermogenic brown and beige fat demonstrates dynamic changes in structure and function in response to cold and beta-adrenergic stimulation [1].

In order to study the patterning and interactions between different cell types in adipose tissue, it is necessary to visualize cells and structures in the tissue in three dimensions (3D). Until recently, our understanding of adipose tissue morphology derived from analyses of thin sections at relatively high magnification. While two-dimensional (2D) histology has played an instrumental role in our appreciation that fat is indeed a tissue or organ [2], these approaches present a number of limitations. Firstly, since adipose tissue has a flexible, amorphous shape, it is not clear whether observations made from tissue sections are representative of the entire tissue. Secondly, the trajectories and targets of structures like nerve projections and blood vessels are challenging to delineate based on 2D sections. Finally, due to its very high lipid content, it is difficult to obtain consistent serial tissue sections that could be used to generate 3D reconstructions.

In recent years, several methods have been developed to facilitate 3D imaging of whole tissues. These methods rely on tissue clearing, which by removing lipids from tissues, eliminates light scatter artifact occurring at lipid-aqueous interfaces. While these methods, such as CLARITY and iDISCO/iDISCO+, have been mainly applied to brain imaging, these tools have been increasingly adapted to study a variety of other organs [3, 4]. Adipose tissue presents a particular challenge given its high lipid content. We have adapted the iDISCO/iDISCO+ protocol to enable the delipidation of adipose tissue while preserving its structural integrity. This method, which we refer to as Adipo-Clear, uses methanol/dichloromethane-based delipidation to achieve the optical transparency necessary for high resolution 3D imaging [5, 6]. While this approach quenches endogenous fluorescence of reporters such as GFP and RFP, cells and structures can be readily visualized by immunolabeling. This protocol can facilitate the study of tissue level biology including interactions between various cell types during development, normal physiology, and in models of disease [7].

2 Materials

Use Milli-Q or Molecular Biology Grade Water and analytical grade reagents to prepare all solutions. Prepare and store all solutions at room temperature. Follow safety procedures when handling hazardous materials and obey all waste disposal regulations.

• 1. Phosphate-buffered Saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4.

• 2. Fixative solution: 4% paraformaldehyde (PFA) in PBS or 10% neutral buffered formalin (see Note 1).

• 3. PBS with 0.05% NaN₃ (see Note 2).

• 4. B1N buffer: 0.3 M glycine, 0.1% Triton X-100 (v/v), 0.01% NaN₃ (w/v), pH 7 to 7.4. Dissolve 22.6 g glycine in 1000 mL water. Add 1 mL Triton X-100 and 0.5 mL 20% NaN₃ (in water). Mix and adjust pH to between 7 and 7.4 with NaOH.

• 5. 100% methanol (see Note 3).

• 6. Dehydration/rehydration buffer 1: 20% methanol, 80% B1N buffer.

• 7. Dehydration/rehydration buffer 2: 40% methanol, 60% B1N buffer.

• 8. Dehydration/rehydration buffer 3: 60% methanol, 40% B1N buffer. (see Note 4).

• 9. Dehydration/rehydration buffer 4: 80% methanol, 20% B1N buffer. (see Note 4).

• 10. 5% H₂O₂ in methanol: add 1 part of 30% H₂O₂ to 5 parts of 100% methanol.

• 11. Dichloromethane (DCM) (see Note 5).

• 12. Whole mount blocking buffer (PTxwH): 0.1% Triton X-100 (v/v), 0.05% Tween 20 (v/v), 2 μg/mL Heparin, 0.02% NaN₃ (w/v), in PBS. Add 1 mL Triton X-100, 0.5 mL Tween 20, 100 μL 20 mg/mL Heparin (in water), and 1 mL 20% NaN₃ to 1000 mL PBS.

• 13. 25% methanol in water.

• 14. 50% methanol in water.

• 15. 75% methanol in water.

• 16. Dibenzyl ether, also known as benzyl ether (DBE) (see Note 6).

• 17. Agarose.

• 18. 15 mL conical tubes.

• 19. An orbital shaker.

• 20. 2 mL microcentrifuge tubes (see Note 7).

• 21. A dissection microscope.

• 22. Weighing boats (see Note 8).

• 23. A light-sheet microscope compatible with DBE (see Note 9).

• 24. An inverted confocal microscope or a two-photon microscope.

3 Methods

The following method is designed for small tissues (e.g., tissues from lean adult mice). Prolonged incubation times and larger buffer volumes are recommended for large tissues (e.g., tissues from obese mice) (see Note 10).

3.1 Tissue Preparation

• 1. Perform standard intracardiac perfusion with approximately 20 mL ice-cold PBS until all blood is removed from the mouse.

• 2. Switch the perfusate to 20 mL ice-cold fixative solution until the mouse body has significantly stiffened.

• 3. Carefully dissect tissues of interest (see Note 11).

• 4. Post-fix the tissues by putting each dissected fat pad in around 15 mL fixative solution in a 15 mL conical tube. Incubate the tissues at 4 °C overnight with mild shaking.

• 5. Wash tissues 3x in PBS at room temperature, 1 h each time.

• 6. Store tissues temporarily in PBS until further processing. For long-term storage, tissues can be stored in PBS with 0.05% NaN₃ at 4 °C (see Note 12).

3.2 Delipidation and Permeabilization

All of the following steps in this section should be carried out on firmly-packed ice or in the cold room (4 °C) with mild shaking (around 100 rpm) on an orbital shaker (unless indicated otherwise). Use a 2 mL microcentrifuge tube with 1.6 mL of solution for incubation. Make sure the sample can freely move in the microcentrifuge tube during shaking. During buffer switching (steps 2–10), remove buffer from the previous step and use a vacuum aspirator to diminish carryover. All buffers used in this section should be pre-chilled at 4 °C before use.

• 1. Gently remove hair and debris from the fat pad under a dissection microscope (see Note 13). Transfer the sample to a clean 2 mL microcentrifuge tube.

• 2. Incubate the sample in a gradient of methanol in B1N buffer (Dehydration/rehydration buffer 1–4 sequentially). Incubate the sample in each buffer for 30 min (see Note 14).

• 3. Incubate the sample in 100% methanol for 30 min.

• 4. Incubate the sample 3x in DCM, 30 min each time (see Note 15).

• 5. Incubate the sample 2x in 100% methanol, 30 min each time.

• 6. Optional: bleach the sample in 5% H₂O₂ in methanol for 1 h or overnight (see Note 16).

• 7. Incubate the sample in a reverse methanol gradient in B1N buffer (Dehydration/rehydration buffer 4–1 sequentially). Incubate the sample in each buffer for 30 min.

• 8. Incubate the sample in B1N buffer for 30 min at room temperature.

• 9. Incubate the sample in B1N buffer overnight at room temperature.

• 10. Incubate the sample in PTxwH buffer for 1 h at room temperature (see Note 17).

3.3 Whole Mount Immunostaining

All of the following steps in this section should be carried out at room temperature, with mild shaking and protection from light. Use a 2 mL microcentrifuge tube with 1.6 mL solution for incubation. Make sure the sample can freely move in the microcentrifuge tube during shaking.

• 1. Dilute primary antibodies in PTxwH buffer to desired concentrations and mix well (see Note 18).

• 2. Centrifuge the diluted antibody solution at around 20,000 × g for 10 min (see Note 19).

• 3. Apply all but the bottom few μL antibody mixture to the sample.

• 4. Incubate the sample with primary antibodies in the dark for 3–5 days with mild shaking.

• 5. Wash the sample in PTxwH buffer in a series of incubation steps: 5 min, 10 min, 15 min, 20 min, 1 h, 2 h, 4 h, and overnight.

• 6. Dilute secondary antibodies in PTxwH buffer to desired concentrations and mix well.

• 7. Centrifuge the diluted antibody solution at around 20,000 × g for 10 min.

• 8. Apply all but the bottom few μL antibody mixture to tissue.

• 9. Incubate the sample with secondary antibodies in the dark for 3–5 days with mild shaking.

• 10. Wash tissues in PTxwH buffer in a series of incubation steps: 5 min, 10 min, 15 min, 20 min, 1 h, 2 h, 4 h, and overnight.

• 11. Wash tissues 3x with PBS, 30 min each time.

• 12. Gently remove hair and lint from the sample under a dissection microscope.

3.4 Optional: Agarose Embedding (see Note 20)

• 1. Position the sample in a weigh boat. Avoid carryover of excess PBS.

• 2. Make 1% agarose by adding 0.5 g agarose to 50 mL PBS. Heat until homogenization.

• 3. Allow the 1% agarose solution to cool down to around 40 °C (see Note 21). Gently pour the agarose solution to fully cover the sample (see Note 22).

• 4. Allow the agarose to solidify.

• 5. Loosen the agarose block from the weigh boat and transfer the sample to a clean platform.

• 6. Trim the agarose to fit the sample and transfer to a new 2 mL microcentrifuge tube.

3.5 Tissue Clearing

All of the following steps in this section should be carried out at room temperature, with mild shaking and protection from light. Use a 2 mL microcentrifuge tube with 1.6 mL solution for each sample. Make sure the sample can freely move in the microcentrifuge tube during shaking.

• 1. Incubate the sample in a gradient of methanol in water (25%, 50%, and 75%), 30 min for each incubation.

• 2. Incubate the sample 2x in 100% methanol, 30 min each time.

• 3. Incubate the sample in 100% methanol for 2 h or overnight.

• 4. Incubate the sample 3x in DCM, 1 h each time (see Note 23).

• 5. Incubate the sample 3x in DBE, at least 2 h each time (see Note 24).

3.6 Microscopy

• 1. Imaging with a DBE-compatible light-sheet microscope (see Fig. 1 for a representative image).

  • (a) Mount the sample onto a sample holder and immerse the sample in a DBE containing imaging chamber.

  • (b) Image with a 1–2× magnification objective to capture the whole-tissue pattern of markers of interest (see Note 25).

  • (c) Image regions of interest with an objective with high magnification (e.g., 4–12×) to obtain a detailed view of the structures.

• 2. Imaging with an inverted confocal microscope or a two-photon microscope.

  • (a) Position the sample in a glass-bottom imaging chamber slide.

  • (b) Use an objective with a long working distance for thick samples (see Note 26).

Fig. 1

An inguinal subcutaneous fat from a postnatal day 10 mouse processed by Adipo-Clear and immunostained with antibodies targeting tyrosine hydroxylase (TH, green) and platelet endothelial cell adhesion molecule (PECAM1, magenta). A representative 40 micron z-stack is presented

4 Notes

• 1. PFA or formalin are toxic. Handle with care and proper personal protective equipment (PPE). Handle the solutions in a fume hood.

• 2. NaN₃ is a highly toxic chemical. Wear proper PPE and work under a fume hood when preparing and handling concentrated NaN₃ (at 5% or greater).

• 3. Methanol is volatile, highly flammable, and toxic. Avoid inhalation and skin or eye contact.

• 4. Due to saturation, glycine crystals in Dehydration/rehydration buffers 3 and 4 will precipitate. Use the liquid solution for incubation. Try to avoid drawing up the crystals.

• 5. DCM is highly volatile and should be handled under a fume hood. Use glass containers for DCM storage. Petri dishes and serological pipets made from polystyrene are not compatible with DCM.

• 6. DBE is hazardous. Handle it in a fume hood with proper PPE. If gloves are contaminated with DBE, discard the gloves as soon as possible following waste disposal regulations. Clean any DBE spills with 100% ethanol. Store DBE in glass containers. Petri dishes and serological pipets made from polystyrene are not compatible with DBE.

• 7. DCM and DBE are not compatible with polystyrene-made microcentrifuge tubes. Use microcentrifuge tubes made from polypropylene. Do not use microcentrifuge tubes for long-term storage of DCM and DBE.

• 8. The size of weight boat or mold depends on tissue size and imaging chamber size.

• 9. For dipping objectives, make sure to use the ones that are compatible with organic solutions such as DBE. Refer to the manufacturer’s manuals for more detailed instructions.

• 10. For processing large tissues, use 5 mL microcentrifuge tubes with 4 mL buffers for the incubation steps in Subheading 3.2, 3.3, and 3.5. In addition, extend incubation time as suggested in Table 1. For agarose embedding (Subheading 3.4), if the tissues are too large to fit in the weight boats, trim the tissues to smaller sizes.

• 11. Blunt-ended forceps are recommended for dissection. Avoid pinching or squeezing the tissues during dissection. Avoid contaminating the tissues with animal fur.

• 12. Tissues can be stored in PBS with 0.05% NaN₃ at 4 °C for several months to 1 year. Tissue quality may deteriorate following prolonged storage.

• 13. Hair, lint, and debris on tissues can cause shadows during imaging, which imperil imaging quality.

• 14. Tissues may appear to shrink slightly due to dehydration.

• 15. Tissues should sink to the bottom of the tubes at the end of each DCM wash to ensure full delipidation. If they do not, increase incubation time until tissues sink. However, fat pads attached with other types of tissues sometimes do not sink even with prolonged incubation time. In this case, extend tissue incubation time to 1 h for each DCM wash regardless. Once DCM is removed at the end of each incubation, quickly apply fresh buffer from the next wash to avoid sample desiccation.

• 16. Hemoglobin from red blood cells can cause strong autofluorescence. If tissues are not well perfused, bleach with 5% H₂O₂ in methanol to reduce background.

• 17. The protocol can be paused at this step if needed. Delipidated tissues can be stored in PTxwH buffer at 4 °C for several months. However, prolonged storage may harm imaging quality.

• 18. It is recommended to first use small pieces of tissue or methanol-treated tissue sections to validate antibodies of interest before proceeding further.

• 19. Centrifuging antibody mixtures helps to remove antibody precipitates.

• 20. Agarose embedding helps to stabilize sample shape during imaging. This step is necessary for imaging adipose tissue with a light-sheet microscope. However, it is not required for imaging with a confocal or two photon microscope.

• 21. While the agarose is cooling down, gently swirl the solution, so the solution remains homogenous. Avoid exposing samples to excessively hot solution.

• 22. Avoid any air bubbles near the embedded sample.

• 23. Tissues should sink to the bottom of the tubes at the end of each DCM incubation step. If not, increase incubation time until tissues sink. Samples can be incubated in DCM overnight.

• 24. Samples should appear transparent in visible light at the end of clearing. After clearing, samples should be stored in the dark at room temperature until imaging. However, long-term storage of the samples at this step is not recommended. Image the samples within a month.

• 25. Avoid secondary antibodies conjugated to fluorophores with short wavelengths due to strong tissue autofluorescence. However, previous studies have shown that tissue autofluorescence signals can outline fat cell contour and reveal overall tissue structures [5]. The extracellular matrix of adipose tissue is mainly composed of collagen, which has a typical emission spectrum ranging from 400 to 550 nm [8, 9]. To capture the tissue autofluorescence, image with a 488-laser line and collect emitted light at a wavelength within the collagen emission spectrum.

• 26. Avoid direct contact between samples and objectives that are not compatible with DBE.

Table 1 Incubation time for large tissues