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...

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 "athlete's paradox"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 "athlete's paradox," 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="Protocol scheme for tissue sample preparation and analysis; includes cryosectioning and microscopy." class="xfigimg" src="/files/ftp_upload/63718/63718fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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 µm 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><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;nbsp;</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 (&amp;oslash; 20 mm, 3 mm thick)&amp;nbsp;</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&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</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&amp;nbsp;mm, &amp;oslash; 150&amp;nbsp;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&amp;nbsp;</td><td></td></tr><tr><td>Microscope Cover Glasses (24x50 mm)</td><td>Assistent&amp;nbsp;</td><td>40990151</td><td></td></tr><tr><td>Microscope Slide Boxes&amp;nbsp;</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&amp;nbsp;</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&quot;), No. 3</td><td>Aesculap</td><td>BB073R</td><td></td></tr><tr><td>Stainless Steel Cup 10oz&amp;nbsp;</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 &quot;Rat teeth tweezers&quot;</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>&lt;strong&gt;Reagents&lt;/strong&gt;</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 &amp;micro;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 &amp;micro;g/mL</td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>ThermoFisher</td><td>D1306</td><td>Used at a final concentration of 0.5 &amp;micro;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. &lt;strong&gt;CAUTION&lt;/strong&gt;: 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>&lt;strong&gt;CAUTION&lt;/strong&gt;: 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>&lt;strong&gt;CAUTION&lt;/strong&gt;: 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>&lt;strong&gt;CAUTION&lt;/strong&gt;:&amp;nbsp; 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&amp;nbsp;</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:&amp;nbsp; 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:&amp;nbsp; 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&amp;nbsp;</td><td>P36930</td><td></td></tr><tr><td>Rat anti-Laminin-2 (&amp;alpha;-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&amp;nbsp;</td><td>4583</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</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 ...

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

Research

JoVE Journal

Medicine

4.0K 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

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Biochemistry

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfr&#945;-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

The replacement of healthy muscle tissue with intramuscular fat is a prominent feature of human diseases and conditions. This protocol outlines how to visualize, image, and quantify intramuscular fat, allowing the rigorous study of the mechanisms underlying intramuscular fat formation.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build ...

Developmental Biology

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 ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Biology

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

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

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

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

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

Confocal Microscopic Analysis of Rat Muscle Mitochondria

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions.
Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).&#160;The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

Author Spotlight: Mitochondrial Remodeling in Skeletal Muscle 

Here, we present a protocol to analyze changes in mitochondrial density and longitudinal distribution by live-skeletal muscle imaging using confocal microscopy for mitochondrial network scanning.

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...