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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Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Murine Adipose Tissue Excision

Shave the abdominal and inguinal regions of two sedated adult male Sprague Dawley rats. Disinfect the shaved regions with an iodine solution. Make a medial longitudinal incision from the sternum to the testicular region, Using a pair of sterile forceps, remove the adipose tissue from the epididymidis and the kidneys.Place the tissue in conical tubes containing 20 milliliters of cold, sterile PBS-AA solution.

Intramuscular Adipose Tissue (IMAT) Visualization with Oil Red O Staining

Prepare the ORO solution by dissolving 0.5 grams of ORO powder in 100 milliliters of isopropanol to generate a stock solution. Prepare the working solution for all muscles by combining the ORO stock solution and deionized water at a 60 to 40 ratio. Cover the working solution for 10 minutes to allow the particulate to settle.Filter it through a 40 micrometer mesh, followed by a 0.22 micrometer syringe filter. Next, remove the formaldehyde solution from the decellularized muscles in the well plate and wash the muscles three times with an equal volume of PBS. Then, replace the PBS with an equal volume of 60%isopropanol solution and incubate it on the rocking shaker for five minutes.Once done, replace the 60%isopropanol solution with ORO working solution before 10 minutes of incubation on the rocking shaker. Next, replace the ORO working solution with an equal volume of 1%SDS and inspect the SDS solution periodically. Once the solution becomes noticeably pink, replace the SDS solution with fresh 1%SDS.When the solution remains clear for 24 hours, replace the 1%SDS with PBS. Under a stereo microscope at four x magnification, remove any obvious debris or particulate stuck to the outside of the stained muscle. If the staining is satisfactory, showing the bright red spheres floating in a transparent matrix, acquire the staining images using a camera attached to the stereo microscope.Properly decellularized muscles were white and semi-transparent. When decellularized muscles were stained with ORO to visualized intramuscular adipose tissue, or IMAT, IMAT lipid droplets were apparent within the clear muscle structures as red spheres. Healthy mouse hind limb muscles showed a little natural IMAT evidenced by little to no red or positive lipid as compared to the hind limb muscles injected with cardio toxin or glycerol.Incomplete decellularization was identified immediately following initial SDS treatment or after the washout of the ORO staining as semi opaque light pink fibers. Incomplete ORO clearance was identified following ORO washout as a pink or red uniform background rather than distinct fiber lines.