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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/63996/63996fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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 &amp;micro;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 &amp;micro;M working solution in dH&lt;sub&gt;2&lt;/sub&gt;O</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&amp;alpha; antibody</td><td>R&amp;amp;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

a-guide-to-examining-intramuscular-fat-formation-its-cellular-origin

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.9K Views.  University of Florida College of Medicine. Een belangrijke barrière bij het bestuderen van intramusculair vet, een kenmerk van sarcopenie en neuromusculaire ziekten, is effectieve conservering en daaropvolgende visualisatie van adipocyten, vooral in bevroren weefselsecties. Het protocol behoudt adipocytenmorfologie en skeletspiersecties uitgelijnd voor robuuste, rigoureuze en reproduceerbare visualisatie en kwantificering van intramusculair vet. Onze analysepijplijn biedt een kader om nieuwe interventies te valideren om vetvorming bi...

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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Immunology and Infection

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.

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 ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biology

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

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 ...

Single Cell Nuclei Isolation from IMAT for Studying Gene Expression Profiles 

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

Author Spotlight: Deciphering the Cellular Mysteries of Intermuscular Adipose Tissue in Humans

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose depot.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and ...

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

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