Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Summary

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Abstract

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

Introduction

Adipose tissue is an essential organ for energy storage and is characterized by dynamic remodelling and nearly unlimited expansion¹. In addition to energy homeostasis, adipose tissue also plays an essential role in hormone secretion of over 50 adipokines to modulate whole-body metabolic function². Adipose tissue has a diverse architecture comprising of various cell types including mature adipocytes, fibroblasts, endothelial cells, immune cells, and adipocyte progenitor cells³. Recent studies have shown that obesity and other metabolic dysfunction can significantly alter adipose tissue function and its microenvironment, which includes but is not limited to enlargement of adipocytes, infiltration of inflammatory cells (e.g., macrophages), and vascular dysfunction³.

Conventional morphological techniques such as histology and cryosectioning demonstrate several limitations in studying adipose biology such as lengthy chemical processing steps, which can lead to tissue shrinkage and structure distortion^3,4. Furthermore, these 2D techniques are insufficient to observe intercellular interactions exerted by different cell types, as the sections obtained are limited to smaller regions of the entire tissue³. Compared to conventional methods of fluorescent imaging, whole-mount staining does not require additional invasive steps, such as embedding, sectioning, and dehydration; thus, this avoids the problem of diminishing antibody specificity. As such, it is a simple and efficient method for imaging adipose tissue, with better preservation of adipocyte morphology and overall adipose tissue structure⁵. Therefore, whole-mount staining as a quick and inexpensive immunolabeling technique was established to preserve adipose tissue 3D architecture^1,6,7,8.

However, despite the preservation of adipose tissue morphology with use of whole-mount staining, this technique is still unable to visualize inner structures beneath the lipid surface of the tissue. Several recent studies^9,10 have established tissue clearing techniques combined with whole-mount immunolabeling^1,6 to allow for comprehensive 3D visualization within adipose tissue. In particular, dense neural and vasculature networks were visualized in recent studies^9,10,11,12 with 3D volume imaging. Indeed, studying the neural and vascular plasticity of adipose tissue under different physiological conditions is essential to study its biology. Immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) tissue clearing is a process comprised of methanol pre-treatment, immunolabeling, and clearing of tissue opaqueness with organic chemical reagents dichloromethane (DCM) and dibenzyl ether (DBE)^13,14. By making the adipose tissue entirely transparent, a more accurate representation of anatomy within the tissue such as blood vessels and neural fibers can be obtained^9,10. IDISCO+ has advantages in that it is compatible with various antibodies and fluorescent reporters^11,14, and it has demonstrated success in multiple organs and even embryoes¹⁴. However, its main limitation is a long incubation time, in which 18 to 20 days are needed to complete the entire experiment.

Another important application of whole-mount staining is the visualization of cell fate in combination with a lineage tracing system. Lineage tracing is the labelling of a specific gene/marker in a cell that can be passed on to all daughter cells and is conserved over time¹⁵. As such, it is a powerful tool that can be used to determine the fate of a cell's progeny¹⁵. Since the 1990s, the Cre-LoxP recombinant system has become a powerful approach for lineage tracing in living organisms¹⁵. When a mouse line that expresses Cre, a DNA recombinase enzyme, is crossed with another mouse line expressing a reporter that is adjacent to a loxP-STOP-loxP sequence, the reporter protein is expressed¹⁵.

For whole-mount staining, the use of fluorescent multicolor reporters is suitable for imaging of adipose tissue because it allows for minimal interference with intracellular activities of the adipocyte¹⁶. However, traditional reporters typically stain the cytoplasm, making it difficult to trace the lineage of white adipocytes, which have limited cytoplasmic content¹⁷. To overcome this problem, the use of membrane-bound fluorescent tdTomato/membrane eGFP (mT/mG) reporter marker is an ideal tool. Membrane-targeted tdTomato is expressed in Cre-negative cells¹⁸. Upon Cre excision, a switch to the expression of membrane-targeted eGFP occurs, making this reporter suitable for tracing the lineage of adipocyte progenitors^17,18 (Supplementary Figure 1).

The purpose of this paper is to provide a detailed protocol for whole-mount staining and show how it can be combined with other techniques to study the development and physiology of adipose tissue. Two examples of applications described in this protocol are its use with 1) multicolor reporter mouse lines to identify various origins of adipocytes and 2) tissue clearing to further visualize the neural arborization in white adipose tissue (WAT).

Protocol

All experimental animal protocols were approved by the Animal Care Committee of The Center for Phenogenomics (TCP) conformed to the standards of the Canadian Council on Animal Care. Mice were maintained on 12-h light/dark cycles and provided with free access to water and food. 7 month old C57BL/6J male mice were used in the whole-mount staining experiment.

NOTE: Sections 1 to 2 are in chronological order, with section 3 being an optional step right after section 1. Section 4 can be performed to analyze adipocyte size and blood vessel density after the completion of section 2.

1. Materials Preparation and Tissue Isolation

1. Prepare fresh 1% paraformaldehyde (PFA) diluted in 1x phosphate buffered saline (PBS) for tissue fixation. Prepare 0.3% nonionic surfactant diluted in 1x PBS (hereafter referred to as PBS-0.3T) for subsequent tissue washing steps.
   NOTE: For each tissue, prepare approximately 1.5 mL of 1% PFA to ensure complete immersion. Fixation volume may be increased or decreased depending on the tissue size.
   CAUTION: This step is hazardous, as PFA is corrosive and toxic. Wear personal protective equipment (e.g., nitrile gloves, lab coat, footwear, safety goggles) and handle in a fume hood.
2. Euthanize animals according to an approved procedure (e.g., cervical dislocation and/or carbon dioxide asphyxiation). Without delay, dissect the desired adipose tissue depots (e.g., inguinal white adipose tissue or perigonadal white adipose tissue)¹⁸.
3. With dissection scissors, cut the tissue on a 100 x 15 mm² Petri dish into pieces approximately 0.5–1 cm in size, and immerse them in microcentrifuge tubes filled with 1% PFA. Keep on ice.

2. Whole-mount Staining of White Adipose Tissue

1. After dissection is complete, move the tissue samples in 1% PFA from the ice to room temperature (RT) for 1 h, and then transfer the tissues to a 12- or 24-well cell culture plate for quicker washing.
2. Wash the tissues with PBS-0.3T, 3 times for 5 min each in RT on a shaker tilted at 22°, with 20–25 tilts per min as the speed.
   NOTE: Use this tilt and speed for all subsequent steps involving the use of a shaker.
   NOTE: If using a multicolor reporter mouse line such as mT/mG and additional antibody staining is not needed, the tissue is ready for microscopy after step 2.2.
3. Add 0.5–1 mL blocking buffer (5% animal serum diluted in PBS-0.3T). Put the plate on the shaker and incubate for 1 h in RT.
4. Aspirate the blocking solution and add primary antibodies diluted in PBS-0.3T with 1% animal serum.
5. Place the plate on a shaker at 4 °C overnight.
6. The next day, wash the tissues with PBS-0.3T 3 times for 5 minutes each in RT.
7. Use appropriate secondary antibodies diluted in PBS-0.3T. Add 0.5–1 mL secondary antibody solutions to each well. Wrap the plate in aluminum foil and incubate it on a shaker for 1 hour at RT.
   NOTE: Dilute the secondary antibody in the dark to prevent photobleaching.
8. After secondary antibody incubation, wash with PBS-0.3T twice for 5 min, each in RT. Image the samples if a neutral lipid stain is not desired. If visualization of lipid droplets is needed using the neutral lipid stain, wash with 1x PBS (without non-ionic surfactant) twice, for 5 min each.
9. After the washing steps, incubate with the neutral lipid stain diluted with 1:1500 dilution factor in 1x PBS for 30 min in RT. The tissues are now ready for microscopy. For future imaging, the tissues can be stored in this solution in 4 °C.
   NOTE: Imaging quality decreases over time; hence, the best time for imaging is within 1 or 2 days.
10. Using forceps, lay the tissue flat on a 24 x 60 mm² glass coverslip and place it on an inverted confocal laser microscope system.
11. If staining of the nuclei with DAPI is desired, add 1–2 drops of mounting medium containing DAPI to completely submerge the tissue and prevent it from drying.
12. To obtain images of whole-mount stained tissues at multiple focal planes, perform Z-stacks of 100–150 μm in depth with 4–6 μm step-size at the desired magnification.

3. Tissue Clearing and Immunolabeling Using iDISCO+

NOTE: This protocol is based on previously published procedures^9,10,19.

1. Fixation and methanol pre-treatment
   1. Incubate the tissues in 4% PFA diluted in 1x PBS at 4°C overnight in 2 mL microcentrifuge tubes.
      NOTE: Leave the tissues in the 2 mL tubes for all the following treatments until imaging.
   2. The next day, wash the tissues in 1x PBS three times, for 1 hour each on a shaker at RT.
      NOTE: This step can be a pausing point to leave the sample overnight at RT or 4 °C.
   3. Dehydrate the tissues at RT in 20%, 40%, 60%, and 80% methanol, subsequently, for 1 h each. Dehydrate in 100% methanol at RT for 1 hour, then transfer the tissues to fresh 100% methanol and incubate at 4 C for 1 h.
      NOTE: Dilute methanol in distilled water. During methanol incubation, there is no need to put samples on a shaker as long as the tissue samples are immersed.
      CAUTION: This step is hazardous, as methanol is toxic. It is highly flammable upon open flames. Wear personal protective equipment (e.g., nitrile gloves, lab coat, safety goggles) and handle in a fume hood. Store the methanol away from ignition and in a flammable safety cabinet.
   4. Bleach the tissues with 5% hydrogen peroxide (H₂O₂; 1 volume of 30% H₂O₂ diluted in 5 volumes of 100% methanol) overnight at 4 °C.
      CAUTION: 30% hydrogen peroxide is very hazardous upon skin and eye contact. Wear personal protective equipment (e.g., nitrile gloves, lab coat, safety goggles) and handle in a fume hood.
   5. Rehydrate the tissues at RT in 80%, 60%, 40%, and 20% methanol and 1x PBS, subsequently, for 1 hour each.
   6. Wash with 0.2% nonionic surfactant diluted in 1x PBS twice, for 1 h each on a shaker at RT.
2. Immunolabeling
   NOTE: Fill up the 2 mL tubes containing the tissue to the top of the tube with the solution used in each step to prevent tissue oxidation as soon as immunolabeling begins, until clearing is completed.
   1. Permeabilize the tissues by incubating them in a solution of 1x PBS, 0.2% nonionic surfactant, 20% dimethyl sulfoxide (DMSO), and 0.3 M glycine at 37 °C on an incubated tabletop orbital shaker for 2 days.
      NOTE: The maximum incubation time for 37 °C permeabilization step is 2 days.
   2. Block the tissues in a solution of 1x PBS, 0.2% nonionic surfactant, 10% DMSO, 5% donkey serum, and 1% Fc block at 37 °C on an incubated tabletop orbital shaker for 2 days.
      NOTE: The maximum incubation time for the blocking step is 2 days.
   3. Incubate the tissues in primary antibodies of interest in a solution of 1x PBS, 0.2% polysorbate 20, 10 µg/mL heparin, 5% DMSO, and 5% donkey serum at 37 °C on an incubated tabletop orbital shaker for 4 days.
   4. Wash with 1x PBS, 0.2% polysorbate 20, and 10 µg/mL heparin on a shaker at RT five times, each for 1 h.
      NOTE: This step can be a pause point to leave samples overnight in RT.
   5. Incubate tissues with secondary antibody in a solution of 1x PBS, 0.2% polysorbate 20, 10 µg/mL heparin, and 5% donkey serum at 37 °C on a tabletop orbital shaker for 4 days.
      NOTE: From step 3.2.5, all samples need to be wrapped with aluminum foil to prevent photobleaching of secondary antibody.
   6. Wash tissues in a solution of 1x PBS, 0.2% polysorbate 20, and 10 µg/mL heparin on a shaker in RT five times, for 2 h each.
      NOTE: This step can be a pausing point to leave the samples overnight at RT.
3. Tissue clearing of white adipose tissue and volume imaging
   1. Dehydrate the tissues contained in 2 mL tubes by incubating in 20%, 40%, 60%, and 80% methanol, subsequently, each for 1 hour at RT. Then, dehydrate the samples in 100% methanol twice at RT.
      NOTE: This step can be a pausing point to leave your samples in 100% methanol overnight at RT.
   2. Incubate the tissues with a mixture of 2 volumes of DCM to 1 volume of methanol for 3 h at RT on a shaker.
      NOTE: DCM is volatile. Make sure the tubes are tightly sealed to prevent evaporation.
      CAUTION: This step is hazardous. DCM is toxic upon inhalation. Prolonged exposure can potentially cause chemical burns. Wear personal protective equipment (e.g., nitrile gloves, lab coat, footwear, safety goggles). Use a fume hood.
   3. Incubate the tissues in 100% DCM twice, for 15 min each on a shaker at RT.
   4. Incubate in 100% DBE in RT until imaging and for sample storage. Before imaging, invert the tubes several times to mix the solutions.
      NOTE: Completely fill tubes with DBE to prevent oxidation, which can result in unsuccessful tissue clearing. Do not shake the tubes during DBE incubation.
      CAUTION: This step is hazardous. DBE is toxic. It can cause irritation to the eyes and skin. Wear personal protective equipment (e.g., nitrile gloves, lab coat, safety goggles) and handle in a fume hood.
   5. Image the whole tissue sample with a light microscope that matches the refractive index of organic solvent DBE. Perform Z-stacking at a desired magnification and step-size for the whole tissue.

4. Examples of Data Analysis from Whole-mount Stained Tissue Images Using ImageJ

NOTE: See https://imageJ.nih.gov/ij/download.html for download and installation instructions.

1. Quantification of blood vessel density (Supplementary Figure 2)
   1. Import the saved images of only the channel with blood vessel immunostaining onto ImageJ.
      NOTE: The images for quantification should be consistent in terms of staining procedure and image acquisition condition. Identical reagents should be used. The exposure time, intensity, and magnification should also be equivalent in the imaging process²⁰.
   2. Convert the image color into black-and-white for blood vessel density quantification. To do so, under “Image” tab, select the “Adjust” command, then the “Color Threshold” option. In the “Threshold Color” display box, choose “Dark Background”²⁰ and select “B&W” under “Threshold Color”.
   3. To measure the percentage of area of blood vessel signal against background, select the “Analyze” tab, then the “Analyze Particles” command. Under the display of “Analyzed Particles”, select the “Clear Results” and “Summarize” options. Click “OK”.
   4. Copy and paste the value of the percentage area under the summary tab into a spreadsheet for analysis. The percentage of area will indicate the blood vessel density. Repeat steps 4.3.1–4.3.4 for each individual image.
2. Quantification of adipocyte size²¹ (Supplementary Figure 3)
   1. Import the saved images of the adipocytes onto ImageJ.
   2. To set the scale of the image, measure the length of the scale in pixels by tracing a line to the scale with a known distance on the image using the straight-line selection tool. Under the “Analyze” tab, select the “Set Scale” command. The distance of the line that was traced before will be automatically calculated in pixels.
   3. The “Set Scale” display box will appear. Enter the known distance and unit of length. Select “Global” to apply the scale setting for all imported images and click “OK”.
   4. To choose area as the method of measurement, under the “Analyze” tab select the “Set Measurements” command. A list of different options for measurements will appear. Select the “Area” option and click “OK”.
   5. Using the freehand selection tool, trace the perimeter of each adipocyte of interest. Under the “Analyze” tab, select the “Measure (Ctrl + M)” command, and the area of the adipocyte will appear. Repeat this procedure for other adipocytes in the image.
      NOTE: To ensure accurate measurements, use multiple images for quantification.
   6. Copy and paste the area measurements into a spreadsheet for further data analysis.

Results

Due to the fragility of adipose tissue, methods involving multiple processing steps and sectioning can lead to disfigurement of adipose tissue morphology³ (Figure 1A). However, whole-mount staining can preserve the morphology of adipocytes, ensuring accurate interpretation of results (Figure 1B).

Over-fixation of adipose tissue leads to fixative-induced autofluorescence. As shown in Figure ...

Discussion

Although conventional techniques such as histology and cryosection offer benefits for observing intracellular structure, whole-mount staining provides a different perspective in adipose tissue research, which enables 3D visualization of cellular architecture of minimally processed tissue.

In order to successfully perform whole-mount staining, the following suggestions should be taken into consideration. Different adipose tissue depots can yield various immunostaining results; thus, the type of...

Materials

Name	Company	Catalog Number	Comments
LipidTox	Life Technologies	H34477
PECAM-1 primary antibody	Millipore	MAB1398Z(CH)
TH (tyrosine hydroxylase) primary antibody	Millipore	AB152, AB1542
DAPI stain	BD Pharmingen	564907
Nikon A1R confocal microscope	Nikon		Confocal microscope
Ultramicroscope I	LaVision BioTec		Light sheet image fluorescent microscope
Alexa Fluor secondary antibodies	Jackson ImmunoResearch		Wavelengths 488, 594 and 647 used
Purified Rat Anti-Mouse CD16/CD32	BioSciences	553141
Dichloromethane	Sigma-Aldrich	270997
Dibenzyl-ether	Sigma-Aldrich	33630
Methanol	Fisher Chemical	A452-1
30% Hydrogen Peroxide	BIO BASIC CANADA INC	HC4060
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650
Glycine	Sigma-Aldrich	J7126
Heparin	Sigma-Aldrich	H3393
Lectin kit I, fluorescein labeled	VECTOR LABORATORIES	FLK-2100
F4/80	Bio-Rad	MCA497GA
VECTASHIELD Hard Set Mounting Medium with DAPI	VECTOR LABORATORIES	H-1500
Paraformaldehyde (PFA)
Phosphate Buffer Saline (PBS)
Triton-X
Tween
Animal serum (goat, donkey)