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 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...

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

3.7K vistas.  Université Catholique de Louvain. Este protocolo permite la cuantificación de las gotas lipídicas y el análisis de su morfología y distribución subcelular de forma específica al tipo de fibra. Esta técnica permite el procesamiento simultáneo de diferentes músculos, ahorrando tiempo y evitando artefactos que podrían ocurrir cuando se procesan de forma independiente. También permite la automatización de la cuantificación de las gotas lipídicas.La miosteatosis se evaluó en los músculos marinos, pero este p...