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

Summary

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.

Abstract

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.

Introduction

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 exercise^5,6. 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 fitness^10,11. 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 action^8,10; 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 ones^8,9,13. 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 signaling^9,15. 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) fibers^8,15,16. 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 specific^17,18. 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 Bodipy^23,24,25, 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 specific^16,25,26,27. 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.

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. Please click here to view a larger version of this figure.

Protocol

All procedures conducted on mice were approved by the Ethical Committee for Animal Experimentation from the Medical Sector at Université Catholique de Louvain (2019/UCL/MD/013).

1. Dissection and preparation of the samples for freezing

1. Label a 3 mm thick piece of cork for each pair of muscles.
2. 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 serve as support (Figure 2B).
   NOTE: The size of the rectangular plastic piece will depend on the size of the muscle. Here, the described dimensions are adapted to the size of the soleus (~9 mg, 1 cm L, 2-3 mm W) and EDL (~5 mg, 1 cm L, 2-3 mm W) of a 3-month-old C57BL/6J male mouse.
3. At the time of dissection, remove the soleus and EDL of the mouse hind limb. To prevent the samples from drying out during dissection, place them on a compress lightly moistened with saline solution in a Petri dish placed on ice.
   NOTE: See Wang et al.²⁸ for explanations on how to dissect these two limb muscles.
4. Place a small drop of Optimal Cutting Temperature compound (OCT) at the cork/plastic junction, avoiding air bubbles.
5. Eliminate excess moisture from the samples by gently drying them with a paper towel (Figure 2A) and place both muscles on the plastic perpendicular to the cork (Figure 2C).
6. Check the orientation of the muscle myofibers under a stereo microscope (Figure 2D).
   NOTE: It is important not to cover the muscle with OCT, since its insulating effect would prevent rapid freezing and will produce freezing artifacts.

2. Freezing skeletal muscle samples for cryosectioning

CAUTION: Freezing of the muscle must be done under a chemical hood, wearing appropriate personal protective equipment (see the Table of Materials).

1. Use a stainless-steel tumbler (~8 cm H, 6 cm Ø) with two side straps attached to it at least 25 cm long (Figure 2F), and fill the tumbler up to 2/3 of its capacity with isopentane.
2. Grabbing the tumbler by the straps, immerse it gently in a polystyrene box filled with liquid nitrogen so that the level of nitrogen outside the container is above the level of isopentane inside (Figure 2F,G).
   CAUTION: When the tumbler comes into contact with the nitrogen, the thermal shock may cause swirling. Ensure that the tumbler is sufficiently immersed but avoid the entry of nitrogen as this would cause isopentane precipitation. If this happens, let the isopentane cool down, fill the tumbler with new isopentane and start over.
3. When the inside of the tumbler is completely covered with a white solid layer of isopentane, take it out of the liquid nitrogen box (Figure 2H).
   NOTE: The melting temperature of isopentane being -159 °C, the edges of the tumbler will turn white when it is cold enough.
4. Gently stir the solid isopentane pieces into the remaining liquid isopentane with forceps until the entire volume becomes liquid again.
5. Re-immerse the tumbler in the liquid nitrogen until isopentane forms white pebbles all over the bottom and edges of the tumbler (Figure 2G,H).
   NOTE: This second cooling step ensures the appropriate freezing temperature of the isopentane.
6. Remove the tumbler from the liquid nitrogen and quickly dip the muscles in the bottom of the tumbler, holding the cork with rat tooth tweezers. Swirl the cork for 15 s in the isopentane and store it at -80 °C until processing (Figure 2I).
   ​NOTE: For short-term storage, the samples can be maintained in a -20 °C freezer. The complete protocol for rapid freezing of skeletal muscle has already been published elsewhere. For detailed references and troubleshooting, see: Meng et al.²⁹, Kumar et al.³⁰, and Leiva-Cepas et al.³¹.

3. Cryosectioning

1. Bring the samples into the chamber of a cryostat previously cooled to -20 °C and the blade temperature set to -25 °C.
   NOTE: Transport samples from the -20 °C/-80 °C freezer to the cryostat in a polystyrene box filled with dry ice and allow the samples to equilibrate for at least 20-30 min to the chamber temperature before cutting.
2. Remove the plastic from the cork with fine tweezers and place the cryostat specimen disc on the quick freeze plate to cool. Once the plate has reached -50 °C, place some OCT on the disc and quickly place the cork on top of the disk, pressing firmly. Wait until the OCT solidifies and the cork is well fixed on the disc.
3. Place the disc on the object head in the desired cutting orientation (Figure 2J,K) and trim the muscle block beyond at least 1/3 of the length of the muscles and until both cross sections of the muscles are visible.
4. Set the cutting thickness to 10 µm and place one section on an adhesion slide to check the correct orientation of the fibers on a brightfield microscope.
   NOTE: Checking the transverse orientation of the fibers is critical. If the fibers are not adequately oriented, adjust the angle of the object head, cut another section and check again.
5. Place two serial cross-sections on two prelabeled adhesion slides: one slide to determine fiber types, the other to quantify lipid content (Figure 2L).
   ​NOTE: Additional serial cross sections can be obtained and kept at -80 °C for other histological studies. However, to avoid altering the lipid content and intracellular morphology²⁴, it is essential to process the first two slides immediately after cutting to prevent air-drying. Freezing and thawing the slides for LD quantification would have the same effect and is thus highly inadvisable.

4. Fiber typing and Bodipy staining

1. Immunohistochemical detection of muscle fiber type
   NOTE: For the following protocol, a total solution volume of 250 µL is sufficient to cover the entire muscle section surrounded by a circle drawn with a hydrophobic pen the approximate size of a 1 cent coin.
   1. Surround the sections with an outline drawn with a hydrophobic pen and rinse with ice-cold 0.1 M phosphate-buffered saline (PBS) for 1 min at room temperature (RT). Place the slide in a humid chamber and block for 90 min at 37 °C in blocking solution (10% normal goat serum (NGS) and 1:30 mouse on mouse (MOM) blocking reagent in PBS).
   2. Remove the blocking solution and incubate the slides for 90 min at 37 °C with the solution containing the primary antibodies (5% NGS, 1:30 MOM blocking reagent, the mouse primary antibodies to recognize type I (IgG2b, at 1:10), type IIa (IgG1, at 1:10), and type IIx (IgM, at 1:5) fibers and a rat anti-laminin (α2 chain, 1:1,000) in PBS.
   3. Wash the slides with PBS 3 x 5 min at RT.
   4. Incubate the slides in the dark for 1 h at RT with the solution containing the secondary antibodies (goat anti-IgG2b AF405 (1:500), goat anti-IgG1 AF488 (1:500), goat anti-IgM AF568 (1:1,000), and goat anti-laminin AF647 (1:500) in PBS).
      CAUTION: For the rest of the protocol, make sure to keep the slides away from light to preserve the fluorescence.
   5. Wash again 3 x 5 min in PBS, rinse in double-distilled H₂O, remove excess water, and mount with an antifade reagent.
      NOTE: Store the slides at 4 °C, protected from light to preserve the fluorescence until image acquisition. Since the slides are not fixed, it is recommended to use freshly made solutions for each experiment, autoclave the PBS, and acquire the images as soon as possible to avoid contamination of the sections.
2. Staining of LDs with Bodipy
   NOTE: Similar to step 4.1., a total solution volume of 250 µL is sufficient to cover the entire muscle section surrounded by a circle drawn with a hydrophobic pen the approximate size of a 1 cent coin.
   1. Surround the sections with an outline drawn by a hydrophobic pen and rinse with ice-cold 0.1 M PBS for 10 min at RT. Use ice-cold PBS for all the rinses and washes.
   2. Fix with cold 4% paraformaldehyde (PFA) without methanol for 10 min at RT. After a first quick rinse, wash the slides with PBS 3 x 5 min at RT.
      CAUTION: Perform this step under a chemical hood.
   3. Place the slide in a humid chamber and block for 1 h at RT with 5% NGS in PBS.
   4. Incubate the slides for 90 min at 37 °C with the solution of the primary antibody (2% NGS and rat anti-laminin (α2 chain, 1:1,000) in PBS). Wash the slides with PBS 3 x 5 min at RT.
      CAUTION: For the rest of the protocol, make sure to keep the slides away from light to preserve the fluorescence.
   5. Incubate for 1 h at RT with the secondary antibody solution containing goat anti-rat-AF647 antibody (1:500) in PBS. Wash the slides with PBS 3 x 5 min at RT.
   6. Incubate for 20 min at RT with a solution of 4',6-diamidino-2-phenylindole (DAPI, 0.5 µg/mL) and BODIPY (1 µg/mL) in PBS.
      NOTE: To prepare BODIPY stock solution, dissolve it in DMSO at a concentration of 1 mg/mL. Different Bodipy formulations are commercially available for LD staining. Depending on the choice made, the staining method is the same (same steps, concentration, and incubation time); however, the acquisition method will be slightly different.
   7. After a first quick rinse, wash the slides with PBS 3 x 5 min at RT, rinse in double-distilled H₂O, remove excess water, and mount with an antifade reagent.
      NOTE: Store the slides at 4 °C, protected from light, to preserve the fluorescence until image acquisition.

5. Acquisition of images

NOTE: Once the staining protocols have been completed, it is important to proceed immediately to image acquisition (within the following 24 h), not only to avoid contamination but also to preserve the morphology, size, and number of LDs.

1. Acquisition of images to assess the fiber-types of each muscle sampled
   NOTE: This step could be achieved with a whole-slide fluorescence scanning microscope or with a conventional epifluorescence microscope. With the latter, manual or automated stitching of the images must be done to reconstruct the section.
   1. For fiber-type recognition, use an epifluorescence microscope with a 10x/0.3 objective. Select excitation filters for DAPI (405 nm), FITC, TRITC, and Cy5 to detect type I, IIa, IIx fibers, and laminin, respectively.
      NOTE: Type IIb fibers will not be immunolabeled. They will be recognized as fibers stained by laminin on the limits of the sarcolemma with a black sarcoplasm.
   2. Adjust the appropriate exposure time for each channel.
   3. When using a conventional epifluorescence microscope, always acquire the images of the entire muscle following the same order to facilitate muscle reconstruction. Make sure that the fibers on the right edge of one image also appear on the left edge of the following image. The same applies for the top and the bottom parts of the images (Figure 3A).
      NOTE: As a reference, for a section of the EDL or the soleus dissected from a 3-month-old mouse, an average of six and eight images, respectively, will cover the entire muscle cross-section.
   4. After the muscle is scanned, upload the captured digital images into any image-processing software for reconstruction (stitching), based on the fiber morphology (laminin) and the histology of the muscle section, and save it as a TIFF, PNG, or JPG file with all the channels (colors) merged (Figure 3A).
2. Acquisition of images with laminin and Bodipy co-staining
   NOTE: To recognize the fiber type and have an estimate of the number of fibers for each type captured, it is essential to have the fiber-type section already scanned and the muscles reconstructed before starting the image acquisition of Bodipy-laminin (Figure 3B).
   1. For Bodipy image observation and acquisition, use a confocal microscope with a 40x oil immersion objective lens with a numerical aperture of 1.4.
   2. Use the following settings: pinhole at 1 AU, 2,048 pixel x 2,048 pixel resolution, pixel size at 0.08 µm, unidirectional mode, scan speed at 4 (~4 µs/pixel), line averaging set to 4x, and digital zoom set to 1.
   3. To avoid cross-talk between Bodipy-558/568 and laminin-AF647, use the sequential scan mode on the confocal software.
      NOTE: When the dye chosen is Bodipy-493/503, simultaneous confocal laser scanning is possible with no crosstalk between the Bodipy channel and laminin-AF647 channel. This will speed up the acquisition of images.
   4. Excite Bodipy-493/503 using the 488 nm laser line or argon laser line, and excite Bodipy-555/568 using the 561 nm diode laser line. Finally, detect laminin-AF647 with a 640 nm diode laser line.
      CAUTION: Bear in mind that Bodipy molecules are very sensitive to photobleaching, so avoid unnecessary laser scanning. To recognize fibers, use only the laser for laminin.
   5. Depending on the dye chosen, set the emission ranges at 570-650 nm for Bodipy-493/503²⁴ and at 565-620 nm for Bodipy-558/568. Set the emission range for laminin at 656-700 nm.
   6. Set the gain and digital gain appropriately so no saturated pixels are detected on the range indicator. Correct the background signal by adjusting the offset.
      NOTE: Filter selection and other above-mentioned scanning parameters must be optimized for each confocal microscope. It is important that all the above-mentioned settings are kept constant for all the images captured from samples to be compared.
   7. To identify the type of fiber among those visualized on the confocal microscope, use a personal laptop on which the image of the section reconstructed after fiber-type immunodetection is checked (Figure 3B).
   8. Once a group of fibers is correctly identified, acquire the image with the Bodipy and laminin channels.
      ​NOTE: It is recommended to note the Bodipy-laminin image name on the region of the muscle where these fibers are located on the image acquired for MyHC recognition to facilitate the later fiber-specific analysis of LDs.

6. Analysis of images

1. Analysis of the fiber-types on each muscle sample
   1. In Fiji (or ImageJ)³², open the TIFF, PNG, or JPG file with the reconstructed muscle obtained from the merge of all the channels used to detect the fiber isoforms.
   2. To start the Cell Counting tool, click on Plugins | Analyze | Cell counter | Cell counter.
   3. On the Cell counter window, click on Actions | Initialize.
   4. Under Counters in the same window, select Type 1.
   5. On the Fiji main window, select the Wand tool.
   6. To quantify the number of fibers of each type, click on each fiber of the same type, so that the program records the number of fibers clicked.
   7. Once finished, select the next type of fiber and repeat the same steps.
   8. When all fibers of the image have been assigned to a given fiber type, click on Results on the Cell Counter window to display the results in a table.
   9. Save this table as a spreadsheet table by clicking on File | Save As on the Results window.
   10. Save and reload the selections on the same image at any time by clicking on Save Markers or Load Markers, respectively, on the Cell Counting window.
2. Analysis of lipid droplets in a fiber-type-dependent manner
   1. Analyze the images of Bodipy and laminin obtained on the confocal using Fiji for the quantification of LDs.
      NOTE: The authors designed a customized macro to automatize the analysis. This macro, along with a step-by-step explanation on how to use it, is available as Supplemental File 1 and Supplemental File 2, respectively.
   2. Open each image with the aid of the Bio-Formats Importer from Fiji. Under the View Stack with option, select Hyperstack | Color mode, Default. Make sure that the Autoscale window is selected.
      NOTE: The following steps will describe the protocol for the analysis of one fiber on the image, but it must be repeated as many times as the number of entire fibers appear on the image.
   3. Use the freehand selection tool to manually select the sarcolemma of the fiber based on the laminin channel (Figure 4A) and press T on the keyboard to record the selection or Region of Interest (ROI) on the ROI window.
   4. Go to Fiji's main window and click on Analyze | Set Measurements, and on the pop-up window, select Area and Feret's diameter. Leave the remaining boxes unchecked and other parameters as they appear by default.
   5. Click on Measure on the ROI window to obtain the Area and Minimal Feret's Diameter (MF) of the fiber selected, and note them for later use.
      NOTE: When analyzing LDs, it is important to have in mind that their size and density vary between the center and the periphery (subsarcolemmal region-SS) of the fiber. Therefore, the analysis must be done separately.
   6. Calculate the value of 1/6 of the MF to delimit the central part of the fiber.
      NOTE: In the macro, the default MF value is set to 6, which means that the reduction applied will be set as 1/6 of the MF. This value was chosen based on the empirical data obtained from the soleus of animals fed a high-fat diet. However, each researcher must change this number based on the empirical data and the muscle analyzed, the type of fiber, and the animal condition.
   7. 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.
   8. On Fiji's main window, click on Edit | Selection | Enlarge and introduce the previously calculated value (from step 6.2.6.) with a minus sign before the number and click on OK. On the ROI window, click on Add[t] (a third ROI must appear) and Delete, to delete the second ROI.
      NOTE: The researcher can check the results by clicking on the Show All box on the ROI window. At this point, two ROIs must appear, one that surrounds the entire perimeter of the fiber (Figure 4B) and another one that is placed in the center (Figure 4C).
   9. Select both ROIs on the ROI window and click on More | XOR | Add[t]. Wait for a third ROI to appear, which corresponds to the periphery of the fiber (Figure 4D).
   10. Save the ROIs by clicking on More | Save to save the ROIs in case a later reanalysis of the same fibers is needed.
   11. Select the Bodipy channel and open the Threshold tool by clicking on Image | Adjust | Threshold on Fiji's main window.
   12. On the Threshold pop-up window, set the values to 70/255, select Yen | B&W method, and click on Dark background | Apply.
       NOTE: The values applied on the Threshold may vary depending on the conditions of the experiment and the threshold must be appropriately set to optimize the analysis. A B&W window with the Bodipy signal above the threshold limit shown in white and the background in black must appear (compare the original Bodipy image in Figure 4E with the one in Figure 4F).
   13. Go to Fiji's main window and click on Analyze | Set Measurements and on the pop-up window, select Area, Area fraction, and Limit to threshold. Leave the remaining boxes unchecked and other parameters as they appear by default.
       NOTE: If the researcher wants to analyze the "circularity" of LDs, which is an index of the spherical morphology that ranges from 1 for a perfect sphere to 0 for a line, click on the Shape descriptors box of the Set Measurements pop-up window.
   14. Go to the ROI window and select the first ROI. On Fiji's main window, click on Analyze | Analyze particles tool.
       NOTE: This tool quantifies the number, size, area covered, and percentage of the total area covered by particles on each selection and saves the results as a spreadsheet file.
   15. Set the values from 2 to Infinity (2-Infinity) on the Analyze Particles window, check the Pixels box, maintain the default circularity values, select Summarize, and click on OK.
       NOTE: To check the results on the fiber, under the Show option, select any of the available options. To have the information of each LD recognized on the selection in a table, check the Display results option on the Analyze Particles window. The results from the analysis of the total area of the fiber are averaged and summarized in a table with several columns (Count, Total Area, Average size, % Area; these correspond to the number of particles [LDs], the area occupied by these particles, their average size, and the percentage of the total area of the selection occupied by particles, respectively). To calculate the density, divide the number of particles by the total area of each selection.
   16. To obtain the values of the center and the periphery of the fiber, repeat steps 6.2.14 and 6.2.15, selecting the second (center) and the third ROI (periphery) each time.
   17. Save the results by clicking on File | Save as on the Summary window.
       NOTE: Include the type of fiber, the condition, and the image name on the assigned name of the results to facilitate later unification and statistical analysis of the data. To analyze the rest of the fibers in the same image, repeat steps 6.2.3 to 6.2.17. For the statistical analysis, at least 10-15 fibers of each type must be analyzed per animal.

Results

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 ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
AxioCam 506 mono 6 Mpix camera	Zeiss
AxioCam MRm 1.4MPix CCD camera	Zeiss
Chemical hood	Potteau Labo	EN-14175
Confocal microscope	Zeiss	LSM800
Cork discs (ø 20 mm, 3 mm thick)	Electron Microscopy Sciences	63305
Cryo-Gloves	Tempshield	16072252
Cryostat	Thermo Scientific	Microm Cryo Star HM 560
Dissecting Stereo Microscope SMZ745	Nikon
Dry Ice
Dumont Forceps	F.S.T	11295-10
Epifluorescence microscope	Zeiss	AxioImage-Apotome Z1
Extra Fine Bonn Scissors	F.S.T	14084-08
FisherBrand Disposable Base Molds (0.7 x 0.7 cm)	ThermoFisher	22-363-552	Used to cut a piece to hold the muscle on the cork for freezing
Glass petri dish (H 25 mm, ø 150 mm)	BRAND Petri dish, MERK	BR455751	Used to place the muscles on ice during dissection
ImmEdge Hydrophobic barrier PAP Pen	Vector Labs	H-4000	Used to create an hidrophobic barrier around the muscle sections
Incubator	MMM Medcenter	Incucell 707
Microscope Cover Glasses (24x50 mm)	Assistent	40990151
Microscope Slide Boxes	Kartell	278	Used as humid chambers for immunohistochemistry
Neck holder	Linie zwo	SB-035X-02	Used as strap to hold the stainless steel tumbler
No 15 Sterile Carbon Steel Scalpel Blade	Swann-Morton	0205
Paint brushes	Van Bleiswijck	Amazon B07W7KJQ2X	Used to handle cryosections
Permanent Marker Pen Black	Klinipath/VWR	98307-R	Used to label slides
Pierce Fixation Forceps	F.S.T	18155-13
Polystyrene Box			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
Scalpel Handle, 125 mm (5"), No. 3	Aesculap	BB073R
Stainless Steel Cup 10oz	Eboxer	B07GFCBPFH	Tumbler to fill with isopentene for muscle freezing
Superfrost Ultra Plus slides	ThermoFisher	J1800AMNZ
Surgical tweezers 1/2 teeth	Medische Vakhandel	1303152	Also called "Rat teeth tweezers"
Vannas Spring Scissors - 3 mm Cutting Edge	F.S.T	15000-00
Weighing boats	VWR international	611-2249
Whole-Slide Scanner for Fluorescence	Zeiss	Axio Scan.Z1
Reagents
Alexa Fluor 405 Goat Anti-Mouse IgG2b	Sigma-Aldrich	SAB4600477	Used at a final concentration of 1:500
Alexa Fluor 488 Goat Anti-Mouse IgG1	ThermoFisher	A-21121	Used at a final concentration of 1:500
Alexa Fluor 568 Goat Anti-Mouse IgM	Abcam	ab175702	Used at a final concentration of 1:1,000
Alexa Fluor 647 goat anti rat-IgG (H+L) secondary antibody	ThermoFisher	A-21247	Used at a final concentration of 1:500
BODIPY-493/503 (4,4-difluoro-1,3,5,7,8-pentametil-4-bora-3a,4a-diaza-s-indaceno)	ThermoFisher	D3922	Used at a final concentration of 1 µg/mL
BODIPY-558/568 C12 (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)	ThermoFisher	D3835	Used at a final concentration of 1 µg/mL
DAPI (4',6-diamidino-2-phenylindole)	ThermoFisher	D1306	Used at a final concentration of 0.5 µg/mL
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D-8418	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.
Formaldehyde solution 4%, buffered, pH 6.9	Sigma-Aldrich	1004969011	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.
Isopentane GPR RectaPur	VWR international	24872.298	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.
Liquid Nitrogen			CAUTION:  Extremely cold. Wear gloves. Handle slowly to minimize boiling and splashing and in well ventilated areas. Use containers designed for low-temperature liquids.
Mouse on mouse Blocking Reagent	Vector Labs	MKB-2213-1	Used at concentration of 1:30
Myosin heavy chain Type I (BA-D5-s Primary Antibody) Gene: MYH7, monoclonal bovine anti mouse IgG2b	DSHB University of Iowa	BA-D5-supernatant	Used at a final concentration of 1:10
Myosin heavy chain Type IIA (SC-71-s Primary Antibody) Gene:  MYH2, Monoclonal bovine anti mouse IgG1	DSHB University of Iowa	SC-71-supernatant	Used at a final concentration of 1:10
Myosin heavy chain Type IIX (6H1-s Primary Antibody), Gene:  MYH1, Monoclonal rabbit anti mouse IgM	Developmental Studies Hybridoma Bank, University of Iowa	6H1-supernatant	Used at a final concentration of 1:5
Normal Goat Serum (NGS)	Vector Labs	S-1000
PBS 0.1 M			Commonly used on histology laboratories
ProLong Gold Antifade Mountant	Invitrogen	P36930
Rat anti-Laminin-2 (α-2 Chain) primary antibody (monoclonal)	Sigma-Aldrich	L0663	Used at a final concentration of 1:1,000
Tissue-Tek O.C.T compound	Sakura	4583
Software
Adobe Illustrator CC	Adobe Inc.		Used to design the figures
Adobe Photoshop	Adobe Inc.		Confocal software
BioRender	https://biorender.com/		Used to design the figures
Fiji/ImageJ	https://imagej.net/software/fiji/		Used to analyse the acquired images
Microsoft PowerPoint	Microsoft		Used to reconstruct the histology of the whole muscle after scanning the fiber types
Zen Blue 2.6	Zeiss		Used to reconstruct the histology of the whole muscle after scanning the fiber types