Name: fiber type and subcellular-specific analysis of lipid droplet content in skeletal muscle
Uploaded: 2022-06-08T13:40:00
Description: Watch this Scientific Journal Video about Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle at JoVE.com

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.

All procedures conducted on mice were approved by the Ethical Committee for Animal Experimentation from the Medical Sector at Universit&#233; Catholique de Louvain (2019/UCL/MD/013).
1. Dissection and preparation of the samples for freezing
<ol>
	<li>Label a 3 mm thick piece of cork for each pair of muscles.</li>
	<li>Through a small incision made with a blade on the center of the cork, insert perpendicularly a rectangular piece of rigid plastic (0.5 cm W, 1 cm H) that will ...

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...

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.

The protocol described herein provides an efficient method to easily quantify LDs in a fiber type and subcellular-specific manner. It shows how, by freezing together two muscles of similar size, such as the EDL and the soleus, the time and resources spent on the following steps are reduced by half.
A complete protocol is provided for immunostaining, image acquisition, and analysis of the different MyHC isoforms expressed in adult mouse muscles. This protocol is based on the one first designed ...

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 ...

This protocol allows the quantification of lipid droplets and the analysis of their morphology and subcellular distribution in a fiber type specific manner. This technique enables the simultaneous processing of different muscles, saving time and avoiding artifacts that could occur when processed independently. It also permits the automatization of lipid droplets quantification.Myosteatosis was assessed in marine muscles, but this protocol could be translated to humans in different conditions, such as obesity, aging, and cancer. Recognizing the same fibers in parallel section could be difficult, but remarkable structures such as axon tweaks or muscle spindles are good landmarks that will help the researcher locate the same fibers on both slides. To begin place two serial cryo frozen cross sections of the muscle specimen on two pre-labeled adhesion slides.One slide to determine fiber types and the other to quantify lipid content. For immunohistochemical detection of muscle fiber type, surround the sections with an outline drawn with a hydrophobic pen. Place it in a humid chamber, and rinse with ice cold 0.1 molar PBS for one minute at room temperature.Next, remove the PBS, add the blocking solution to the slide, and incubate for 90 minutes at 37 degrees Celsius. Later remove the blocking solution and incubate the slides for 90 minutes at 37 degrees Celsius with the solution containing the primary antibodies. Wash the slides thrice with PBS for five minutes each, at room temperature.Add the solution containing the secondary antibodies and incubate the slides in the dark for one hour at room temperature, hereafter ensure to keep the slide away from the light and proceed with the three washes in PBS for five minutes each, then rinse the slide in double distilled water, after removing the excess water, mount the slide with antifade reagent and store it at four degrees Celsius protected from light. To stain the lipid droplet with bodipy, surround the muscle section with an outline drawn by a hydrophobic pen before rinsing it with ice cold 0.1 molar PBS for 10 minutes. Both slides must be stained simultaneously.Fix the section with cold four percent paraformaldehyde without methanol for 10 minutes at room temperature. After first quick rinse wash the slides thrice with PBS for five minutes. Block the slide with 5%normal goat serum in PBS for one hour in human chamber.Followed by incubating the slide with the solution of the primary antibody comprising 2%normal goat serum and rat anti-laminin in PBS for 90 minutes. After washing slides thrice in PBS, carry out the next incubation with the secondary antibody solution containing goat, anti-rat, Alexa fluor 647 antibody in PBS at room temperature for one hour. Make sure that the slide is protected from the light.After incubation wash the slide thrice with PBS. Next, incubate the section for 20 minutes with dapi and bodipy solution. After a quick rinse, wash thrice with PBS, and once with double distilled water, then remove the excess water and mount the section with an anti-fade reagent.Once the muscle is scanned, upload the captured digital images into any image processing software for reconstruction. Based on the fiber morphology and the histology of the muscle section, save the image in a suitable format with all the channels merged. For bodipy image observation and acquisition, set the parameters for a confocal microscope, with a 40 X oil immersion objective lens and a numerical aperture of 1.4, as described in the manuscript.To avoid crosstalk between bodipy and 558, 568, and laminin Alexa fluor 647;Use the sequential scan mode on the confocal software. Excite bodipy and 493, 503 using the 488 nanometer laser line or argon laser line, and excite bodipy and 555, 568 using the 561 nanometer diode laser line. Finally, detect laminin Alexa fluor 647 with a 640 nanometer diode laser line, depending on the dye chosen, set the emission rates at 570 to 650 nanometers for bodipy and 493, 503.And at 565 to 620 nanometers for bodipy and 558, 568. Set the emission range for laminin at 656 to 700 nanometers. Next, set the gain and digital gain appropriately to avoid detecting saturated pixels on the range indicator.Correct the background signal by adjusting the offset. To identify the type of fiber on the confocal microscope, use a laptop on which the image of the previously reconstructed slide with the fiber type immuno detection can be checked. Once a group of fibers is correctly identified acquire the image with the bodipy and laminin channels.Open each image with the aid of the bioformat's importer from Fiji. Under the view stack with option select hyper stack, then color mode and default. Make sure that the auto scale window is selected.Use the free hand selection tool to manually select the sarcolemma of the fiber based on the laminin channel. And press T on the keyboard to record the region of interest on the ROI window. Go to Fiji's main window and click on analyze and set measurements.Then on the popup window select area and for raised diameter, leave the remaining boxes unchecked and other parameters as they appear by default. Click on measure on the ROI window to obtain the area and minimal for raised diameter of the fiber selected and note them for later use. Calculate the value of one sixth of the minimal for raised diameter to delimit the central part of the fiber.Click on the ROI window, Click on add to have a duplicate of the first ROI and select the second ROI that appears on the window. On Fiji's main window, click on edit, then selection and enlarge. To introduce the previously calculated value with a minus sign before the number and click on okay.On the ROI window, click on add, A third ROI must appear, and delete to delete the second ROI. On the ROI window select both ROIs and click on more then ZOR and add, wait for a third ROI to appear which corresponds to the periphery of the fiber. If re-analysis of the same fibers is needed, save the ROIs by clicking on more then save.Select the bodipy channel and open the threshold tool by clicking on image, adjust, and threshold tabs on Fiji's main window. On the threshold popup window set the values to 70 and 255. Then select YEN and black and white method.Before clicking on dark background. Finally hit the apply tab. Go to Fiji's main window and click on analyze and set measurements.On the popup window, select area, area fraction and limit to threshold. Leave the remaining boxes and parameters as default. Go to the ROI window and select the first ROI.On Fiji's main window click on analyze, then analyze particles tool. On the analyzed particles window set the values from two to infinity and check the pixels box. Maintain the default circularity value and select summarize before clicking okay.To obtain the values of the center and the periphery of the fiber, repeat the selection of the second and the third ROI each time. Then save the results by clicking on file, then save as in the summary window. Include the type of fiber on the name of the file.This immunohistochemical protocol permits the recognition of mice, slow oxidative fibers, like type 1 and 2A, fast glycolytic fibers, such as type 2X and 2B, And the presence of hybrid fibers. By measuring the area and minimal for raised diameter of the muscle fiber a size dependent reduction was applied to obtain the central and the peripheral areas of each fiber. Remarkable structures of each muscle section, such as axon twigs or muscle spindles are good landmarks that could help locate the same fibers on both sides.The settings described for acquiring bodipy laminin staining by confocal microscopy. Permitted, the visualization and analysis of the size, number, and distribution of lipid droplets from three to six fibers simultaneously. The method also allowed the quantification of three important parameters, namely the percentage of the fiber area occupied by the lipid droplets, the density, and the average size of lipid droplets.Improper freezing procedure and failure to achieve the right temperature of isopentane resulted in an ice crystal formation inside the muscle fibers. When sections were not aligned transverse the lipid droplet quantification and comparison between fibers, muscles, or animals could not be performed. The processing of the second slide must be done simultaneously with the first.Air drying the slide after cryosection for 15 minutes will significantly reduce the size and number of lipid droplets. Bodipy can be used to study the interaction of lipid droplets with other subcellular organelles, or protein stack with fluorescence of a different spectrum, or with genetically modified GFP models. Myosteatosis has been reported to be a poor prognostic factor for several diseases.Therefore, this technique could be used to monitor the progression of a disease or to evaluate the effect of a treatment.

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

Research

JoVE Journal

Medicine

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 ...

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.

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

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 ...

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.

Do&#039;s and Don&#039;ts in the Preparation of Muscle Cryosections for Histological Analysis

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 ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.