This work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS-Cr&#233;dit de Recherche J.0022.20) and the Soci&#233;t&#233; Francophone du Diab&#232;te (SFD-Roche Diabetes Care).C.M.S. is the recipient of a Ph.D. fellowship from the FRIA (FNRS). M.A.D.-L.d.C. received a fellowship from the Wallonie-Bruxelles International Excellence Program.
The authors thank Alice Monnier for her contribution to the development of this protocol and Caroline Bouzin for her expertise and technical help in the image acquisition process. We also thank the 2IP-IREC imaging platform for access to the cryostat and the microscopes (2IP-IREC Imaging Platform, Institute of Experimental and Clinical Research, Universit&#233; Catholique de Louvain, 1200 Brussels, Belgium). Finally, the authors would like to thank Nicolas Dubuisson, Romain Versele, and Michel Abou-Samra for constructive criticism of the manuscript. Some of the figures of these article were created with BioRender.com.

The authors have no conflicts of interest to declare.

The protocol detailed here describes an efficient method to quantify LDs tagged with Bodipy on a fiber-type- and subcellular-specific basis. In recent years, classical lipid dyes, such as ORO or Sudan Black B, have been substituted with a new array of cell-permeable, lipophilic, fluorescent dyes that bind to neutral lipids (e.g., Bodipy). Available as different conjugates, Bodipy has been proven very effective at tagging LDs to study their morphology, dynamics, and interaction with other organelles, not only in different fixed tissues and cells23,38,39,40 but also in living cells41,42.
Within the protocol, there are several critical aspects that the researcher must consider to obtain optimal results. It is essential to place the chosen muscles correctly on the cork to ensure perfect alignment and orientation of muscle myofibers, since once frozen, only slight readjustments of the holder can be made on the cryostat. Moreover, it is critical to start the immunostaining protocol immediately after the sections are cut, since air-drying cryosections (for only 15 min) has severe adverse effects on LDs, resulting in a 66% decrease in LD density and a 37% decrease in the average size24. Similarly, freezing and thawing the slides would have adverse effects and are therefore not recommended. A third important feature of this protocol is related to the fixation of samples. For the identification of the different MyHC isoforms with the antibodies cited here, tissue fixation must be avoided as it disrupts the binding of the chosen antibodies to their epitopes. If the muscle samples have been previously fixed, the reader is referred to a recently published paper using different antibodies43. Only PFA without methanol is recommended for the labeling and quantification of LDs, because the use of methanol or acetone disrupts the morphology of LDs23,44. Furthermore,a permeabilization step is not necessary for the quantification of LDs on slides immunolabeled with laminin. Since some groups have shown that permeabilization with detergents such as TritonX-100, saponin, or glycine can reduce the size or number of LDs44,45, permeabilization is highly discouraged. Finally, the fine-tuning of the confocal microscope settings is crucial to recognize only the neutral lipids present in LDs and not those in membranes of other organelles24.
Here, Bodipy-558/568 C12 was used as a LD marker. However, the most frequently Bodipy used for tagging LDs and studying their morphology and location within the skeletal muscle myofibers is Bodipy-493/503. Both dyes are similar in their incubation time, working concentration, and their narrow emission spectra, although their excitation and emission ranges are slightly different. For a complete protocol on the use of Bodipy-493/503, the reader is referred to the work of Listenberger and Brown46 or Spangenburg et al.23. Bodipy-558/568 C12 has been used for staining LDs in other tissues such as adipose tissue47, degenerating retina48, fibrotic kidneys49, or fibroblasts50. This Bodipy yields a similar staining to the one obtained with ORO (see Supplemental Figure S1) but with considerably less technical burden and more specificity24,51. Moreover, Bodipy-558/568 C12 has the advantage of enabling the quantification of LDs in combination with the detection of proteins or organelles tagged with a green-spectrum secondary antibody (see Supplemental Figure S2 and Yan et al.49) or with GFP-genetically modified models52. These two applications are very powerful tools to unravel the dynamics and interactions of LDs with other cell organelles and proteins. Nevertheless, when no colabeling of proteins within the cells is intended, the authors recommend the use of the best-characterized LD dye Bodipy-493/503.
One of the limitations of this protocol is related to the acquisition of images and the quantification of LD morphological characteristics. Technological advances in confocal laser scanning microscopy have greatly improved the plane resolution compared with wide-field microscopes and permit 3D reconstruction of objects when scanning the sample in the Z plane. This has been very useful for the study of LD morphology24 and interaction with other proteins in skeletal myofibers38,53, but has increased the image acquisition and processing time, since each 3D-reconstructed image is composed of several independent 2D images. In the above protocol, confocal images were acquired with a 40x objective lens and only in 2D. This method allows the acquisition of three to six myofibers per image instead of only one as it would be when using a 63x objective. This results in lower resolution and an overall estimation of the LD size and morphology that is not completely accurate. Nevertheless, it permits the analysis of a greater number of fibers per sample, which is also an important fact to consider, especially in animal experiments, in which a high number of muscles and conditions must be compared.
Here, it is shown that, by freezing two muscles of similar size that are adjacent to each other34, the processing time of the samples is considerably decreased, and possible artifactual differences derived from their independent processing are reduced. Moreover, this method of analyzing LDs takes into consideration the differences between the subcellular distribution of LDs within and among fiber types54. Selecting the perimeter of the myofiber and reducing this selection in relation to the fiber&#39;s Minimal Feret&#39;s diameter facilitates the study of the size and density of central and peripheral (subsarcolemmal) LDs independently. Applying this method is extremely important since it has been demonstrated that the subsarcolemmal accumulation of LDs directly contributes to insulin resistance. While some reports in humans have studied LDs in a fiber type- and location-dependent fashion11,16,25, this is the first time, to our knowledge, that this method has been applied to muscles from rodents. Moreover, the quantification of LDs with the aid of a self-designed macro for Fiji significantly reduced and simplified the image analysis time. Both the macro and a step-by-step explanation on how to use it are available as Supplemental File 1 and Supplemental File 2, respectively.
Overall, the authors consider that the protocol described herein could be a useful tool for other researchers studying lipid metabolism in skeletal muscle. The techniques explained may be applied to different muscles and under distinct conditions (fasted/overfed, trained/sedentary, young/aged, lean/obese) and will hopefully help to better comprehend the dynamics of LDs, the importance of their interactions with other components of the cell, how lipids are stored and metabolized in glycolytic and oxidative fibers, and their role in the onset of insulin resistance in type 2 diabetes.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis is negatively correlated with muscle mass and strength and with insulin sensitivity1. Moreover, recent studies indicate that the degree of myosteatosis could be used as a prognostic factor for other conditions such as cardiovascular disease2, non-alcoholic fatty liver disease3, or cancer4. Lipids can accumulate in skeletal muscle between muscle fibers as extramyocellular lipids or within the fibers, as intramyocellular lipids (IMCLs). IMCLs are predominantly stored as triglycerides in lipid droplets (LDs) that are used as metabolic fuel during physical exercise5,6. However, when lipid supply exceeds demand, or when mitochondria become dysfunctional, IMCLs will be implicated in muscle insulin resistance, as seen in metabolically unhealthy, obese individuals and in type 2 diabetes patients7. Intriguingly, endurance athletes have similar, if not higher, levels of IMCLs to those found in obese patients with type 2 diabetes mellitus, while maintaining high insulin sensitivity. This phenomenon is described as the &#34;athlete&#39;s paradox&#34;8,9, and is explained by a more nuanced appraisal of muscle LDs, related to their size, density, localization, dynamics, and lipid species composition.
First, LD size is inversely correlated to insulin sensitivity and physical fitness10,11. In fact, smaller LDs exhibit a relatively greater surface area for lipase action and, thus, potentially have a greater capacity to mobilize lipids12. Second, LD density (number/surface) plays a controversial role in insulin action8,10; yet, it seems to be increased in athletes. Third, the subcellular localization of LDs is important, since LDs located just below the surface membrane (subsarcolemmal or peripheral) exert a more deleterious effect on insulin sensitivity than central ones8,9,13. The latter provide fuel to central mitochondria, which have a greater respiratory activity and are more specialized to meet the high energy demand required for contraction14. By contrast, peripheral LDs supply subsarcolemmal mitochondria, which are involved in membrane-related processes8. Finally, beyond triglycerides, specific complex lipids within the muscle may be more deleterious than others. For instance, diacylglycerol, long-chain acyl-CoA, and ceramides may accumulate in muscle when the triglyceride turnover rate is low, thereby impairing insulin signaling9,15. Returning to the &#34;athlete&#39;s paradox,&#34; endurance athletes have a high number of smaller central LDs with elevated turnover rates in type I (oxidative) fibers, while obese and diabetic patients have larger peripheral LDs with low turnover rates in type II (glycolytic) fibers8,15,16. In addition to their role in energy storage and release, LDs via derived fatty acids (FA) and a coat protein (perilipin 5) could also function as critical players involved in the transcriptional regulation of FA oxidation and mitochondrial biogenesis8. Because of their crucial implications in physiology and pathophysiology, in-depth studies on LDs dynamics and functions are warranted.
Although there are several techniques to study IMCLs, they are not all suitable to accurately quantify LD size, density, and distribution in a fiber-specific manner. For example, the assessment of IMCLs by magnetic resonance spectroscopy, while being non-invasive, offers a level of resolution that is not sufficient to study the size and precise location of LDs within the fiber, and it is not fiber-type specific17,18. Likewise, biochemical techniques performed on whole-muscle homogenates19 cannot assess the location and size of lipids. Consequently, the most adequate method to analyze LD morphology and location is quantitative transmission electronic microscopy13, but this technique is expensive and time-consuming. Therefore, confocal fluorescence imaging on preparations with dyes such as Oil Red O (ORO)20,21, monodansylpentane (MDH)22, or Bodipy23,24,25, has emerged as the best tool for these studies.
Here, a complete protocol is described, including tissue sampling and processing, Bodipy staining, and confocal image acquisition and analysis to quantify LD size, number, and localization in mouse muscle cryosections. Since IMCLs are not evenly distributed among oxidative and glycolytic fibers, and each fiber type regulates LD dynamics differently, the study of IMCLs must be fiber-type specific16,25,26,27. Therefore, this protocol uses immunofluorescence on serial sections to identify myosin heavy chain (MyHC) isoform(s) expressed by each fiber. Another advantage of this protocol is the simultaneous processing of a glycolytic (extensor digitorum longus, EDL) and an oxidative (soleus) muscle placed side-by-side before freezing (Figure 1). This simultaneous processing not only saves time but also avoids variability due to separate processing of the samples.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63718/63718fig01.jpg" /> 
Figure 1: Schematic overview of the procedure. After muscle dissection (1), similar-size selected muscles are prepared and frozen together (2). Serial transverse sections of 10 &#181;m are obtained using a cryostat and directly mounted on adhesion slides (3). From two serial slides, the first (4A) is immunolabeled for laminin and stained with Bodipy to recognize LDs and the second (4B) is immunostained with antibodies against MyHCs for the recognition of muscle fiber types. Images are acquired using a confocal microscope for Bodipy (5A) and an epifluorescence microscope for muscle fiber types (5B). Images are analyzed in Fiji by applying a threshold and quantifying particles (6A) to obtain the number, average size, density, and percentage of the total area occupied by LDs (7) or counting cells (6B) to obtain the percentage of fibers of each type in the section (7). Abbreviations: LDs = lipid droplets; EDL = extensor digitorum longus; MyHCs = myosin heavy chain isoforms. <a href="https://www.jove.com/files/ftp_upload/63718/63718fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxioCam 506 mono 6 Mpix camera</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxioCam MRm 1.4MPix CCD camera&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical hood</td>
			<td>Potteau Labo</td>
			<td>EN-14175</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cork discs (&#248; 20 mm, 3 mm thick)&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>63305</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-Gloves</td>
			<td>Tempshield</td>
			<td>16072252</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>Microm Cryo Star HM 560</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Stereo Microscope SMZ745</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps</td>
			<td>F.S.T</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioImage-Apotome Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>F.S.T</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>FisherBrand Disposable Base Molds (0.7 x 0.7 cm)</td>
			<td>ThermoFisher</td>
			<td>22-363-552</td>
			<td>Used to cut a piece to hold the muscle on the cork for freezing</td>
		</tr>
		<tr>
			<td>Glass petri dish (H 25&#160;mm, &#248; 150&#160;mm)</td>
			<td>BRAND Petri dish, MERK</td>
			<td>BR455751</td>
			<td>Used to place the muscles on ice during dissection</td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic barrier PAP Pen</td>
			<td>Vector Labs</td>
			<td>H-4000</td>
			<td>Used to create an hidrophobic barrier around the muscle sections</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>MMM Medcenter</td>
			<td>Incucell 707&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses (24x50 mm)</td>
			<td>Assistent&#160;</td>
			<td>40990151</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide Boxes&#160;</td>
			<td>Kartell</td>
			<td>278</td>
			<td>Used as humid chambers for immunohistochemistry</td>
		</tr>
		<tr>
			<td>Neck holder</td>
			<td>Linie zwo</td>
			<td>SB-035X-02</td>
			<td>Used as strap to hold the stainless steel tumbler</td>
		</tr>
		<tr>
			<td>No 15 Sterile Carbon Steel Scalpel Blade</td>
			<td>Swann-Morton</td>
			<td>0205</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brushes</td>
			<td>Van Bleiswijck</td>
			<td>Amazon B07W7KJQ2X</td>
			<td>Used to handle cryosections</td>
		</tr>
		<tr>
			<td>Permanent Marker Pen Black</td>
			<td>Klinipath/VWR</td>
			<td>98307-R</td>
			<td>Used to label slides</td>
		</tr>
		<tr>
			<td>Pierce Fixation Forceps</td>
			<td>F.S.T</td>
			<td>18155-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Box&#160;</td>
			<td></td>
			<td></td>
			<td>H 12 cm x L 25 cm x W 18 cm, used as a liquid nitrogen container and to transport the samples to the cryostat</td>
		</tr>
		<tr>
			<td>Scalpel Handle, 125 mm (5&#34;), No. 3</td>
			<td>Aesculap</td>
			<td>BB073R</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Cup 10oz&#160;</td>
			<td>Eboxer</td>
			<td>B07GFCBPFH</td>
			<td>Tumbler to fill with isopentene for muscle freezing</td>
		</tr>
		<tr>
			<td>Superfrost Ultra Plus slides</td>
			<td>ThermoFisher</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tweezers 1/2 teeth</td>
			<td>Medische Vakhandel</td>
			<td>1303152</td>
			<td>Also called &#34;Rat teeth tweezers&#34;</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 3 mm Cutting Edge</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing boats</td>
			<td>VWR international</td>
			<td>611-2249</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole-Slide Scanner for Fluorescence</td>
			<td>Zeiss</td>
			<td>Axio Scan.Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 405 Goat Anti-Mouse IgG2b</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4600477</td>
			<td>Used at a final concentration of 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG1</td>
			<td>ThermoFisher</td>
			<td>A-21121</td>
			<td>Used at a final concentration of 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Goat Anti-Mouse IgM</td>
			<td>Abcam</td>
			<td>ab175702</td>
			<td>Used at a final concentration of 1:1,000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 goat anti rat-IgG (H+L) secondary antibody</td>
			<td>ThermoFisher</td>
			<td>A-21247</td>
			<td>Used at a final concentration of 1:500</td>
		</tr>
		<tr>
			<td>BODIPY-493/503 (4,4-difluoro-1,3,5,7,8-pentametil-4-bora-3a,4a-diaza-s-indaceno)</td>
			<td>ThermoFisher</td>
			<td>D3922</td>
			<td>Used at a final concentration of 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BODIPY-558/568 C12 (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)</td>
			<td>ThermoFisher</td>
			<td>D3835</td>
			<td>Used at a final concentration of 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>Used at a final concentration of 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D-8418</td>
			<td>Used to solve Bodipy for the 1 mg/mL stock solution. CAUTION: Toxic and flammable. Vapors may cause irritation. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.</td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4%, buffered, pH 6.9</td>
			<td>Sigma-Aldrich</td>
			<td>1004969011</td>
			<td>CAUTION: May cause an allergic skin reaction. Suspected of causing genetic defects. May cause cancer. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.</td>
		</tr>
		<tr>
			<td>Isopentane GPR RectaPur</td>
			<td>VWR international</td>
			<td>24872.298</td>
			<td>CAUTION: Extremely flammable liquid and vapor. May be fatal if swallowed and enters airways. May cause drowsiness or dizziness. Repeated exposure may cause skin dryness or cracking. Wear protective gloves/protective clothing/eye protection/face protection.</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>CAUTION:&#160; Extremely cold. Wear gloves. Handle slowly to minimize boiling and splashing and in well ventilated areas. Use containers designed for low-temperature liquids.</td>
		</tr>
		<tr>
			<td>Mouse on mouse Blocking Reagent&#160;</td>
			<td>Vector Labs</td>
			<td>MKB-2213-1</td>
			<td>Used at concentration of 1:30</td>
		</tr>
		<tr>
			<td>Myosin heavy chain Type I (BA-D5-s Primary Antibody) Gene: MYH7, monoclonal bovine anti mouse IgG2b</td>
			<td>DSHB University of Iowa</td>
			<td>BA-D5-supernatant</td>
			<td>Used at a final concentration of 1:10</td>
		</tr>
		<tr>
			<td>Myosin heavy chain Type IIA (SC-71-s Primary Antibody) Gene:&#160; MYH2, Monoclonal bovine anti mouse IgG1</td>
			<td>DSHB University of Iowa</td>
			<td>SC-71-supernatant</td>
			<td>Used at a final concentration of 1:10</td>
		</tr>
		<tr>
			<td>Myosin heavy chain Type IIX (6H1-s Primary Antibody), Gene:&#160; MYH1, Monoclonal rabbit anti mouse IgM</td>
			<td>Developmental Studies Hybridoma Bank, University of Iowa</td>
			<td>6H1-supernatant</td>
			<td>Used at a final concentration of 1:5</td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td>Vector Labs</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 0.1 M</td>
			<td></td>
			<td></td>
			<td>Commonly used on histology laboratories</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen&#160;</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Laminin-2 (&#945;-2 Chain) primary antibody (monoclonal)</td>
			<td>Sigma-Aldrich</td>
			<td>L0663</td>
			<td>Used at a final concentration of 1:1,000</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T compound</td>
			<td>Sakura&#160;</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator CC</td>
			<td>Adobe Inc.</td>
			<td></td>
			<td>Used to design the figures</td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe Inc.</td>
			<td></td>
			<td>Confocal software</td>
		</tr>
		<tr>
			<td>BioRender</td>
			<td>https://biorender.com/</td>
			<td></td>
			<td>Used to design the figures</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ</td>
			<td>https://imagej.net/software/fiji/</td>
			<td></td>
			<td>Used to analyse the acquired images</td>
		</tr>
		<tr>
			<td>Microsoft PowerPoint</td>
			<td>Microsoft</td>
			<td></td>
			<td>Used to reconstruct the histology of the whole muscle after scanning the fiber types</td>
		</tr>
		<tr>
			<td>Zen Blue 2.6</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used to reconstruct the histology of the whole muscle after scanning the fiber types</td>
		</tr>
	</tbody>
</table>

fiber type and subcellular-specific analysis of lipid droplet content in skeletal muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Watch this Scientific Journal Video about Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle at JoVE.com

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

fiber-type-subcellular-specific-analysis-lipid-droplet-content

Universidad San Sebastian

Research

JoVE Journal

Medicine

3.9K Views.  Université Catholique de Louvain. Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Video: Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...