Name: visualization of 3d white adipose tissue structure using whole-mount staining
Uploaded: 2018-11-17T14:45:00
Description: Watch this Scientific Journal Video about Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining at JoVE.com

This work was funded by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada, Pilot and Feasibility Study Grant of Banting &#38; Best Diabetes Centre (BBDC), the SickKids Start-up Fund to H-K.S., Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning to J-R.K.

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 t...

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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...

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 distortion3,4. 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&#160;and overall adipose tissue structure5. Therefore, whole-mount staining as a quick and inexpensive immunolabeling technique was established to preserve adipose tissue 3D architecture1,6,7,8.
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 studies9,10&#160;have established tissue clearing techniques combined with whole-mount immunolabeling1,6&#160;to allow for comprehensive 3D visualization within adipose tissue. In particular, dense neural and vasculature networks were visualized in recent studies9,10,11,12&#160;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 obtained9,10. IDISCO+ has advantages in that it is compatible with various antibodies and fluorescent reporters11,14, 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&#39;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 progenitors17,18&#160;(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).

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

visualization of 3d white adipose tissue structure using whole-mount staining

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.

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 ...

Watch this Scientific Journal Video about Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining at JoVE.com

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

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.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

This method can help answer key questions in the adipose biology field, such as how intercellular interactions and adipose tissue physiology change upon different metabolic conditions such as fasting and obesity. The main advantage of this technique is that it optimally preserves the original adipose 3D structure and avoids lengthy processing steps that are usually required for conventional immunohistochemistry. Although this method can provide insight into the structure of the adipose tissue, it can also be applied to other organ systems, such as the nervous system, by whole-mount staining the brain.Generally, individuals new to this method will struggle because of the difficulties in getting property tissue sites, finding the proper fixation period, and the optimal antibody concentrations. After dissection is complete, transfer the dish containing the tissue samples in 1%paraformaldehyde from the ice to room temperature for one hour. Then transfer the tissues to either a 12 or 24-well cell culture plate for quicker washing.It's important to remember that the whole-mount staining procedure must begin immediately after dissection and that there are no pause points between each step. Wash the tissues with PBS-0.3T on a shaker tilted at 22 degrees with a speed between 20 and 25 tilts per minute at room temperature for five minutes. Repeat this wash two additional times.After this, add approximately 0.5 to one milliliter of blocking buffer containing 5%animal serum. Incubate the plate on a shaker using the previous conditions for one hour. Aspirate the blocking solution, and add on milliliter of primary antibodies diluted in PBS-0.3T with 1%animal serum to each well.Incubate the plate overnight on a shaker at four degrees Celsius with a tilt of 22 degrees and a speed of 20 to 25 tilts per minute. The next day, use PBS-0.3T to wash the tissues at room temperature for five minutes. Repeat this wash two additional times.Then, add between 0.5 and one milliliter of appropriate and diluted secondary antibody solutions to each well. Wrap the plate in aluminum foil and incubate it on a shaker at room temperature for one hour. After this, wash the plate twice with PBS-0.3T at room temperature for five minutes per wash.If visualization of liquid droplets is needed during the neutral lipid stain, wash twice with 1x PBS with each wash lasting five minutes. Add the neutral lipid stain diluted in PBS, and incubate at room temperature for 30 minutes. To begin imaging, use forceps to lay the tissue flat on a 24 by 60 millimeter glass cover slip.If nuclei staining with DAPI is desired, add one to two drops of a mounting medium that contains DAPI to completely submerge the tissue and prevent it from drying. Then, place the slide on an inverted confocal laser microscope system. To obtain images of whole-mount stained tissue at multiple focal planes, perform Z stacks of 100 to 150 micrometers in depth with step size of four to six micrometers at the desired magnification.In this study, whole-mount staining is used to preserve adipose tissue architecture. Unlike methods that involve multiple processing steps and sectioning, which can lead to disfigurement of the adipose tissue morphology, the whole-mount staining method can preserve the morphology of adipocytes, ensuring accuracy when interpreting results. Overfixation of adipose tissue leads to fixative-induced fluorescence, which can be indicated by overlapping identical regions, such as in the tyrosine hydroxylase and PECAM-1 signals seen here.Properly fixed, whole-mount stain samples, however, show separate and distinct signals, indicating that these signals are not autofluorescence. Imaging whole-mount stained adipose tissue allows for the quantification of morphological changes under different experimental conditions. In particular, adipose tissue is highly vascularized, which is important in mediating metabolic homeostasis upon rapid changes in energy level.For instance, C57 black 6J mice, that undergo 24 hours of fasting, display a significantly smaller adipocyte size, which indicates lipolysis, and a trend in elevated blood vessel density when compared to continuously fed mice. Clearing adipose tissues with iDISCO+enables adipose tissues to become optically transparent. Immunolabeling of adipose tissue that has gone through tissue clearing shows much denser neural arborization compared to that observed in whole-mount staining without tissue clearing at the same magnification.While attempting this procedure, it's important to remember not to overfix your samples for more than a hour at room temperature in PFA, otherwise, this will increase background autofluorescence. However, this fixation period can be prolonged if samples are fixed at four degrees Celsius. Following this procedure, other methods like quantification using ImageJ, can be performed in order to answer additional questions, like how cellular architecture, such as adipocyte sites and blood vessel density, change in response to various physiological conditions.

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