We thank the members of the Kopinke laboratory for helping with data collections and critical reading of the manuscript. We also thank the members of the Myology Institute at the University of Florida for their valuable input on the manuscript. The work was supported by the NIH grant 1R01AR079449. Figure 2 was created with Biorender.

The authors declare no competing interests.

This protocol outlines an extensive and detailed protocol that allows for efficient visualization and rigorous quantification of intramuscular fat. By splitting the same muscle into two parts, one being used for immunofluorescence and the other for RT-qPCR analysis, this protocol is also very versatile. It can also be combined with genetic lineage tracing of FAPs to study their conversion into adipocytes under certain conditions and is highly adaptable to label and image multiple additional cell types.
The most commonly used ways to visualize intramuscular fat are paraffin sections followed by hematoxylin and eosin staining or frozen sections stained for lipophilic dyes such as Oil Red O (ORO). However, while paraffin-processed tissues maintain the best histology, the same process also extracts all lipids preventing the use of lipophilic dyes. Although lipophilic staining methods will work on both PFA-fixed and unfixed tissue sections, lipid droplets are easily displaced by applying pressure to the coverslip, thereby distorting the spatial distribution of intramuscular fat. To circumvent this, a recent study established a rigorous protocol to visualize ORO+ adipocytes using a whole-mount approach. For this, the authors decellularized the TA to visualize the spatial distribution of intramuscular fat throughout the whole TA41. As powerful as this technique is, it also prevents the use of other co-stains to mark additional cellular structures. The whole mount immunofluorescence approach presented here can be used to co-stain adipocytes with a variety of markers allowing for fine mapping of the cellular environment. One major challenge, however, is tissue penetration of the antibodies. The more fibers are kept together, the more difficult it will be for the antibodies to equally penetrate and bind all available antigens. Thus, this method is most effective when looking at small groups of fibers. At the same time, this is also a limitation as the overall anatomical location of intramuscular fat is being lost when focusing on only small, peeled-off fiber bundles. However, with the current development of novel tissue clearing methods plus new imaging technology, greater tissue penetration and visualization will be possible in the future42,43,44.
While prior fixation of muscle tissue preserves adipocyte morphology, it also creates a challenge to assess the size of myofibers, an important measurement of muscle health. Myofiber size is determined by measuring the cross-sectional area of myofibers. We have previously reported that prior fixation of muscle tissue will cause most markers available to outline myofibers to fail31. To overcome this hurdle, we have developed a novel image segmentation pipeline, which allows the measurement of myofiber size even in fixed muscle sections31. Thus, we have established a robust and efficient tissue processing pipeline that, combined with this protocol, overcomes most disadvantages caused by prior fixation of muscle tissue.
Another major advantage of this approach is versatility. By splitting the TA into two parts, the amount of information that can be obtained from one muscle is maximized. This not only reduces animal numbers but also adds an extra layer of control by confirming histology through gene expression and vice versa. In addition, many different genes can be examined beyond adipogenic genes. The isolated RNA can also be used for a whole muscle RNAseq experiment. Finally, the snap-frozen muscle piece can also be used for protein work. One limitation of this protocol is the possibility of the injury not being consistent across the full length of the TA. This could lead to a scenario where the two muscle parts diverge in the amount of intramuscular fat they contain and may warrant exclusion of such a sample from any downstream analysis. It is, therefore, recommended to not simply rely on RT-qPCR to draw major conclusions about the amount of intramuscular fat, but rather as supportive data to the histological quantifications.
Together, this protocol outlines a robust, efficient, and rigorous tissue processing pipeline that will allow visualization and quantification of intramuscular fat, the first step in developing novel treatment options to combat fatty fibrosis. At the same time, it is versatile and can be adapted to many different cell types within the muscle as well as adipocytes in other tissues.

The infiltration of healthy muscle tissue with fatty fibrosis is a prominent feature of Duchenne Muscular Dystrophy (DMD) and other neuromuscular diseases, as well as sarcopenia, obesity, and diabetes1,2,3,4,5,6,7,8,9,10. Although increased fat infiltration in these conditions is strongly associated with decreased muscle function, our knowledge of why and how intramuscular fat forms is still limited. FAPs are a multipotent mesenchymal stromal cell population present in most adult organs, including skeletal muscle11,12. With age and in chronic diseases, however, FAPs produce fibrotic scar tissue and differentiate into adipocytes, which are located between individual myofibers and form intramuscular fat13,14,15,16,17,18,19,20.
To start combating intramuscular fat formation, the mechanisms of how FAPs turn into adipocytes need to be defined. PDGFR&#945; is the &#34;gold-standard&#34; marker in the field to identify FAPs within the muscle of multiple species13,16,17,18,20,21,22,23,24,25,26,27. As a result, several murine tamoxifen-inducible Cre lines, under the control of the Pdgfr&#945; promotor, have been generated, allowing for genetically manipulating FAPs in vivo using the Cre-LoxP system27,28,29. For example, by combining this inducible Cre line with a genetic reporter, lineage tracing of FAPs can be performed, a strategy we have successfully applied to fate map FAPs in muscle and white adipose tissue20,30. Besides lineage tracing, these Cre lines provide valuable tools to study the FAP-to-fat conversion.
One major obstacle in defining the mechanism of the adipogenic conversion of FAPs into intramuscular fat is the ability to rigorously and reproducibly quantify the amount of intramuscular fat that has formed under different conditions. The key is to balance the preservation of muscle and fat tissue and match this with the available staining methods to visualize adipocytes. For example, skeletal muscle is often snap-frozen without prior fixation, preserving myofibers but disrupting adipocyte morphology (Figure 1). In contrast, fixation followed by paraffin embedding, while displaying the best tissue histology, including adipocytes, removes all lipids, thereby rendering most lipophilic dyes, such as the commonly used dye Oil Red O, unusable.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63996/63996fig1v2.jpg" /> 
Figure 1: Representative images of intramuscular fat in snap-frozen versus fixed muscle tissues. (A) Schematic overview of the experimental setup. Immunofluorescent images showing adipocytes (yellow), myofibers (gray), and nuclei (cyan) within both (B) snap-frozen and (C) fixed TAs at 21 days post glycerol injury. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63996/63996fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The protocol described here preserves myofiber and adipocyte morphology and allows visualization, and analysis, of multiple cell types. This approach is based on immunofluorescence staining of adipocytes in paraformaldehyde (PFA)-fixed muscle tissue, which allows for co-staining with multiple antibodies. It can also be easily adapted to spatially display intramuscular fat in intact tissue using whole-mount imaging, thereby providing information on the cellular microenvironment of fat within the muscle. In addition, this protocol can be combined with our recently published approach to determine the cross-sectional area of myofibers in fixed muscle tissues31, an important measurement to assess muscle health. Combining this approach with genetic lineage tracing to fate-map the differentiation of FAPs into adipocytes is also outlined here. Thus, the versatile protocol described here enables rigorous and reproducible assessment of FAPs and their differentiation into intramuscular fat in tissue sections and intact tissues.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63996/63996fig2v2.jpg" /> 
Figure 2: Schematic protocol overview. Schematic overview of tissue processing in which one-third of the TA is removed, snap-frozen, and homogenized for subsequent RNA isolation and transcription analysis via RT-qPCR. The other two-thirds of the TA is PFA-fixed and processed for immunostaining on frozen sections or whole-mount fibers. <a href="https://www.jove.com/files/ftp_upload/63996/63996fig2v2large.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>16% PFA (Pack of 12, 10 mL bottles)</td>
			<td>Electron Miscroscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td>microcentrifuge tubes for snapfreezing/bead beating</td>
		</tr>
		<tr>
			<td>2-Methylbutane (4 L)</td>
			<td>Fisher Chemical</td>
			<td>O3551-4</td>
			<td>isopentane</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 proof)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab fragment donkey anti-mouse</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-007-003</td>
			<td>mouse-on-mouse blocking</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-chicken secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>703-545-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-rabbit secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 donkey anti-goat secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11057</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 donkey anti-rabbit secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phalloidin antibody</td>
			<td>Invitrogen</td>
			<td>A12380</td>
			<td>Dissolved in 1.5 mL methanol (~66 &#181;M working solution)</td>
		</tr>
		<tr>
			<td>BioLite 24-well Multidishes</td>
			<td>ThermoFisher Scientific</td>
			<td>930186</td>
			<td>24 well plate for PFA tissue incubation</td>
		</tr>
		<tr>
			<td>Biometra TOne</td>
			<td>analytikjena</td>
			<td>8462070301</td>
			<td>Thermal cycler</td>
		</tr>
		<tr>
			<td>Chicken anti-GFP antibody</td>
			<td>Aves Labs</td>
			<td>GFP-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform/isoamyl alcohol 24:1(v/v) for molecular biology, DNAse, RNAse, and Protease free</td>
			<td>ThermoFisher Scientific</td>
			<td>AC327155000</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma Aldrich</td>
			<td>C8267</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI stain</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>150 &#181;M working solution in dH2O</td>
		</tr>
		<tr>
			<td>Donkey Serum (100 mL)</td>
			<td>Millipore Sigma</td>
			<td>5058837</td>
			<td>for blocking solution</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>sharp-tipped tweezers</td>
		</tr>
		<tr>
			<td>Fine Scissors Straight 9 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Glycerol, 99.5%, for molecular biology (500 mL)</td>
			<td>Acros Organics</td>
			<td>327255000</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-PDGFR&#945; antibody</td>
			<td>R&#38;D</td>
			<td>AF1062</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>VWR</td>
			<td>230301V</td>
			<td>for Tamoxifen incubation</td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td>hydrophobic pen</td>
		</tr>
		<tr>
			<td>Insulin Syringe with Micro-Fine IV needle (28 G)</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe with Slip Tip, 1 mL</td>
			<td>BD</td>
			<td>329654</td>
			<td>Insulin syringe without needle, for oral gavaging</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>78938441</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMi8 inverted microscope</td>
			<td>Leica</td>
			<td></td>
			<td>micrscope used for widefield IF and confocal imaging</td>
		</tr>
		<tr>
			<td>Micro Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td>positively charged microscope slides</td>
		</tr>
		<tr>
			<td>mouse anti-MYOD antibody</td>
			<td>Invitrogen</td>
			<td>MA1-41017</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-PAX7 antibody (supernatant)</td>
			<td>DSHB</td>
			<td>AB 428528</td>
			<td></td>
		</tr>
		<tr>
			<td>MX35 Premier+ Microtome blades</td>
			<td>ThermoFisher Scientific</td>
			<td>3052835</td>
			<td>microtome blades</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND2000</td>
			<td>spectrophotometer for RNA yield</td>
		</tr>
		<tr>
			<td>Play-Doh</td>
			<td>Hasbro</td>
			<td></td>
			<td>modeling compound</td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>A25742</td>
			<td>green dye PCR master mix</td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Puralube</td>
			<td>17033-211-38</td>
			<td>vet ophthalmic ointment</td>
		</tr>
		<tr>
			<td>QuantStudi 6 Flex Real-Time 384-well PCR System</td>
			<td>Applied Biosystems</td>
			<td>4485694</td>
			<td>qPCR machine</td>
		</tr>
		<tr>
			<td>Rabbit anti-perilipin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9349S</td>
			<td></td>
		</tr>
		<tr>
			<td>Red-Rotor Shaker</td>
			<td>Hoefer Scientific</td>
			<td>PR70-115V</td>
			<td>shaker for IF staining</td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific Slip-Rite Cover Glass</td>
			<td>ThermoFisher Scientific</td>
			<td>152460</td>
			<td>coverslips</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74106</td>
			<td>contains mini spin columns</td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 1.5 ml, natural</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Tubes RB (2 mL)</td>
			<td>QIAGEN</td>
			<td>990381</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Alfa Aesar</td>
			<td>14314</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 2.8 mm</td>
			<td>Precellys</td>
			<td>KT03961-1-101.BK</td>
			<td>small beads</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 5 mm</td>
			<td>QIAGEN</td>
			<td>69989</td>
			<td>medium beads</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 7 mm</td>
			<td>QIAGEN</td>
			<td>69990</td>
			<td>large beads</td>
		</tr>
		<tr>
			<td>Stainless Steel Disposable Scalpels</td>
			<td>Miltex</td>
			<td>327-4102</td>
			<td>scalpel</td>
		</tr>
		<tr>
			<td>Stainless steel feeding tube, 20 G x 38 mm, straight</td>
			<td>Instech Laboratories</td>
			<td>FTSS-20S-3</td>
			<td>gavage needle</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Toronto Research Chemicals</td>
			<td>T006000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Plus O.C.T. Compound</td>
			<td>Fisher HealthCare</td>
			<td>4585</td>
			<td>embedding medium</td>
		</tr>
		<tr>
			<td>TissueLyser LT</td>
			<td>QIAGEN</td>
			<td>85600</td>
			<td>bead beater</td>
		</tr>
		<tr>
			<td>TissueLyser LT Adapter, 12-Tube</td>
			<td>QIAGEN</td>
			<td>69980</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomold</td>
			<td>Sakura</td>
			<td>4566</td>
			<td>specimen molds</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>guanidium thiocyanate</td>
		</tr>
		<tr>
			<td>Tween20 (500 mL)</td>
			<td>Fisher BioReagents</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Micro Slides - Superfrost Plus</td>
			<td>VWR</td>
			<td>48311703</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheaton Coplin staining jars</td>
			<td>Millipore Sigma</td>
			<td>S6016</td>
			<td>Coplin jar</td>
		</tr>
	</tbody>
</table>

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.

Immunofluorescent visualization of intramuscular fat 
Following the steps above and viewing Figure 1A, TA tissue sections were gathered from a 21 day post glycerol injury that were either snap-frozen immediately after harvesting in LN2-cooled isopentane or were fixed in 4% PFA for 2.5 h. After cryosectioning and staining both samples, images were taken at mid-belly, the largest area of the TA. PERILIPIN+ adipocytes from the unfixed TAs (Figure 1B) have significantly altered morphology compared to fixed sections (Figure 1C), making their identification, visualization, and subsequent quantification much more difficult and potentially inaccurate. To note, the first PERILIPIN+ lipid droplets were detected at around 5 days post-injury, with most adipocytes having formed by day 7. By 21 days post-injury, adipocytes had fully matured.
As the amount of fat per TA strongly correlates with the severity of the induced injury, the TAs must be injured significantly to effectively observe and study intramuscular fat formation. Practicing injections using ink into cadaver TAs is a great way to improve injury severity. Successful injuries tend to be above 50% of the muscle. To note, injured areas of the muscle represent areas devoid of muscle fibers or areas that are populated by muscle fibers that contain at least one centrally-located nucleus, a known hallmark of a regenerating muscle fiber.
This protocol can be readily adapted to stain for FAPs and fat in 3D. For this, multiple myofibers from the TA post-fixation were carefully separated, followed by whole-mount immunofluorescence. The key is to properly secure the fibers to the glass slide and, at the same time, to avoid over-compression of the tissue. By using moldable clay feet, the user can adjust the required thickness and secure the coverslip to the slide, even allowing the use of an inverted microscope (Figure 4A). This method was used successfully to label PDGFR&#945;+ FAPs, Phalloidin+ myofibers, and PERILIPIN-expressing adipocytes (Figure 4B, Supplemental Video 1 and Supplemental Video 2). After obtaining images at multiple z-planes spanning up to 150 &#181;m in thickness, the 3D rendering module within the microscope software was used to create a 3D reconstruction.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63996/63996fig4v3.jpg" /> 
Figure 4: Whole-mount immunofluorescent staining. (A) Top and side view of how to mount the sample and add coverslip for whole-mount staining. (B) Representative 3D reconstructions of FAPs (green; left) and adipocytes (red; right) along with myofibers (gray) and nuclei (blue). Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63996/63996fig4v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Quantification of intramuscular fat 
Once images have been taken of intramuscular fat, the Cell Counter function in ImageJ/FIJI was used to manually count the number of PERLIPIN+ adipocytes (Figure 5A). Next, the total area of the muscle section as well as the injured area, defined by centrally-located nuclei within myofibers, was determined. To control for injury severity, the total number of adipocytes was divided by the injured area resulting in the number of fat cells per 1 mm2 of injured muscle. Usually, TAs that display &#60;30% injury are excluded from the quantifications. To note, although adipocytes are rare without injury, ranging from zero to eight per cross-sectional area, the total number of adipocytes are still normalized by total area. As highlighted in Figure 5B, a glycerol injury causes massive amounts of intramuscular fat compared to an uninjured TA muscle. Alternatively, as Perilipin staining is very clean with a high signal-to-noise ratio, it is also possible to use the Analyze Particle function to determine the total area occupied by Perilipin. However, this method will not be able to distinguish between smaller vs. fewer adipocytes. Up to three sections from a minimum of four individual animals were imaged and quantified, and the average number of fat cells present per mouse was reported.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63996/63996fig5v2.jpg" /> 
Figure 5: Quantifications of intramuscular fat. (A) Representative image of how to count PERILIPIN+ adipocytes (white) using the Cell Counter function in ImageJ. Scale bar: 50 &#181;m. (B) Whole TA adipocyte quantifications 21 days post glycerol injection normalized to 1 mm2 of the injured area. Each dot represents the average of one mouse. Error bars shown as SEM. **** = p &#60; 0.0001. (C) RNA layer after homogenization and subsequent phase separation by chloroform is being used for RT-qPCR analysis. (D) Fold changes in expression levels of Pparg and Cepb&#945;, early adipogenic genes, and Plin1 and Adipoq, two mature adipocyte markers, at different time points post glycerol injury. Each dot represents the average of one mouse. Error bars shown as SEM. <a href="https://www.jove.com/files/ftp_upload/63996/63996fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To independently confirm the amount of intramuscular fat present, gene expression levels of various adipogenic markers can be determined. For this, RNA can be isolated from a portion of the same TA muscle used for immunofluorescence (see steps above) at different points post-injury. A bead beater was used in combination with guanidium thiocyanate to homogenize the tissue. After adding chloroform followed by centrifugation, the upper RNA-containing layer was carefully extracted, and mini spin columns were used for RNA cleanup (Figure 5C). This method routinely produces high quality and quantity of RNA suitable for all downstream analyses such as RT-qPCR and RNAseq. For RT-qPCR, the relative expression levels of adipogenic to housekeeping genes were determined, and any relative changes were assessed following the &#916;&#916;CT method38. As described in Figure 5D, compared to uninjured TA muscle, glycerol injury induces expression of early adipogenic markers such as Pparg and Cebp&#945; as soon as 3 days post-injury. Mature markers, such as Adiponectin (Adipoq)&#160;and Perilipin (Plin1), can be detected as early as 5 days after glycerol injury.
Genetic lineage tracing of adipocytes 
The adipocyte staining protocol presented here can be easily adapted to include genetic lineage tracing of FAPs to map and follow their fate into adipocytes. We have, for example, previously demonstrated that recombination could be induced via tamoxifen administration in Pdgfr&#945;CreERT2; Rosa26EYFP&#160;mice 2 weeks prior to the injury, effectively removing the floxed stop coding and indelibly activating EYFP expression in FAPs (Figure 6A). We achieved high recombination efficiencies with the tamoxifen regimen presented here, with ~75% of PDGFR&#945;+ FAPs expressing EYFP20, similar to what other laboratories have reported27,39,40. Demonstrating that FAPs are indeed the cellular origin of intramuscular fat, the majority of FAPs have turned into EYFP+ PERILIPIN-expressing adipocytes 7 days post glycerol injury (Figure 6B).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63996/63996fig6v3.jpg" /> 
Figure 6: Lineage tracing of FAPs. (A) Schematic overview of the experimental setup. (B) Representative immunofluorescent images showing successful recombination and activation of EYFP (yellow) within PDGFR&#945;+ FAPs (red, arrowheads) and PERILIPIN+ adipocytes (red, asterisks). Scale bars: 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63996/63996fig6v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Detection of multiple cell types 
This protocol can also be used to visualize the myogenic compartment. Using antibodies against PAX7 and MYOD1, muscle stem cells (MuSCs) and myoblasts, respectively, can be readily detected 5 days post glycerol injury even in PFA-fixed muscle tissue section (Figure 7). Thus, the presented protocol is versatile and adaptable to not only label and image adipocytes and FAPs but also other cell types of the myogenic lineage.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63996/63996fig7v2.jpg" /> 
Figure 7: Muscle stem cell and myoblast immunofluorescent staining. (A) Schematic overview of the experimental setup. (B) Representative immunofluorescent images showing successful staining of muscle stem cell (MuSC) (yellow, left) with PAX7 and myoblasts (yellow, right) with MYOD1. LAMININ outlines the myofibers (white), and nuclei are in cyan. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63996/63996fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video 1: 3D rendering of FAPs. Three-dimensional reconstruction of myofibers, FAPs, and nuclei stained for PHALLOIDIN (gray), PDGFR&#945; (green), and DAPI (blue), respectively, 21 days post injury. <a href="https://www.jove.com/files/ftp_upload/63996/FAPs.mp4" target="_blank">Please click here to download this Video.</a>
Supplemental Video 2: 3D rendering of intramuscular fat. Volumetric rendering of myofiber bundles (gray, PHALLOIDIN) and intramuscular fat (red, PERILIPIN), which has replaced a myofiber 21 days post glycerol injury. <a href="https://www.jove.com/files/ftp_upload/63996/FAT.mp4" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle at JoVE.com

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

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 ...

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3.7K Views.  University of Florida College of Medicine. 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. 

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.