We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. Tissue Harvest and Freezing of Muscle Tissues
<ol>
	<li>Euthanize the mouse with an overdose of isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane). Perform this step in a fume hood and use a secondary containment method such as a desiccator or a bell jar containing a cotton swab soaked in isoflurane.</li>
	<li>Confirm death by firmly squeezing footpad. Use a secondary method of euthanasia such as cervical dislocation.</li>
	<li>Wet the fur with 70% alcohol to reduce the sticking of loose hair strands on the muscles. Peel the skin off the limb using a fine forceps such as #5.</li>
	<li>Carefully separate the muscle of interest (tibialis anterior (TA) in this case) from the bones underneath, starting from the tendon and remove.</li>
	<li>Excise the muscle, cover it in OCT (Optimum Cutting Temperature compound), and lay it flat on a labeled disposable freezing mold. Make sure that the muscle is at its normal physiological orientation (head to tail) without stretching. Alternatively, freeze the muscle on small styrofoam squares and pin it using insect pins to ensure the correct length of the muscles.</li>
	<li>Chill 2-methylbutane (isopentane) in a steel beaker using liquid nitrogen. This takes on average 3 - 5 min. Keep stirring intermittently until white precipitates start to form on the bottom and walls of the beaker. 
	NOTE: When this occurs, isopentane has reached optimal freezing temperature (-150 &#176;C)</li>
	<li>Dip the molds containing muscles in the chilled 2-methylbutane. Freeze small muscles such as the diaphragm and extensor digitorum longus (EDL) for 6 - 12 sec and larger muscles like the gastrocnemius soleus (GS) and triceps for 15 - 20 sec. Do not freeze the muscles for too long as this may lead to cracking.</li>
	<li>Transfer the frozen tissues on to dry ice and let the isopentane evaporate (note that this process takes on average around 15 - 20 min). Wrap the tissues in aluminum foil and store them at ultra low temperature (-70 &#176;C to -80 &#176;C) until sectioning.</li>
</ol>
2. Embedding the Tissues in OCT to Prepare Tissue Block
<ol>
	<li>Transport tissues on dry ice to cyrostat to prevent thawing. Transfer tissue to cryostat chamber (set at -20 to -24 &#176;C to avoid full thawing of tissues) and let them equilibrate for 30 min.</li>
	<li>Turn the Peltier bay cooling on. If the cryostat does not have this cooling, use aerosol cooling spray to quickly freeze the OCT. Do not let the muscle thaw at any point during the embedding as this can result in freeze damage.</li>
	<li>Add a uniform thin layer of OCT on the specimen disc and let it cool. When most of the OCT is frozen on the bottom while still liquid on the top, insert the tissue at the correct orientation (perpendicular to OCT for transverse sections and parallel to OCT for longitudinal sections). Use aerosol cooling spray to quickly freeze OCT. Touch the unembeded part of the tissue with heat extractor and wait for 45 sec.</li>
	<li>Add another thin layer of OCT around the muscle, spray with aerosol cooling spray and extract the heat as in the previous step.</li>
	<li>Keep adding OCT in small increments and quickly freeze the OCT using a combination of aerosol cooling spray and heat extractor until the whole muscle is covered.</li>
	<li>Put the heat extractor on the top of the tissue block and wait for 5 min.</li>
</ol>
3. Preparing Sections and Identifying the Sections from Mid-belly Region of the Muscles
<ol>
	<li>Mount the specimen onto the specimen head. Tighten the knob to secure the disc. Make sure that the specimen disc is in good contact with the specimen head.</li>
	<li>Adjust the chamber temperature to between -21 and -24 &#176;C and wait until the set temperature is achieved.</li>
	<li>Adjust the thickness settings (7 &#181;m); trim the block by advancing or retracting the block using coarse and fine settings.</li>
	<li>Collect the sections on pre-warmed positively charged microscope slides by pressing the top of the slide onto the sections, using the attraction between opposite charges to facilitate the adherence of the cut sections to the slide.</li>
	<li>Identify the mid-belly sections by measuring cross-sectional area (CSA) of the sections. Do this using imaging software on the computer that is connected to the microscope. 
	NOTE: Phase contrast images are taken and circumference of the entire muscle is measured via a tracing function on the software program. This will yield both CSA as well as minimum ferret diameter (measure of fiber cross-sectional size that is expressed as the smallest distance between two parallel tangents on opposite sides of a particle) measurements. Note: When CSA and minimum ferret diameter measurements begin to plateau, mid belly has been reached.</li>
	<li>Inventory and archive the slides at -80 &#176;C for future staining. 
	NOTE: OCT does not need to be removed prior to staining procedures. Once slides are coverslipped, they are ready to be stained immediately.</li>
</ol>
4. Staining
NOTE: While detailed step-by-step procedures can be found on the product data sheet and should be followed for staining procedures, personal optimization may be required to obtain optimal staining.
<ol>
	<li>Because mounting media (see table) is not miscible with water, dehydrate slides through increasing grades of alcohol (2 min each in 70%, 95%, and 100% ethanol) and clear&#160;them with xylene (100% for 5 min) to ensure slides do not become opaque over time.</li>
	<li>Coverslip sections and let them dry. Image with microscope equipped with imaging software.</li>
</ol>

<ol>
	<li>Bonnemann, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+approach+to+the+congenital+muscular+dystrophies.">Diagnostic approach to the congenital muscular dystrophies.</a> Neuromuscular disorders : NMD. 24, 289-311 (2014).</li><li>Caughey, J. E., Pachomov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diaphragm+in+dystrophia+myotonica.">The diaphragm in dystrophia myotonica.</a> J Neurol Neurosurg Psychiatry. 22, 311-313 (1959).</li><li>Fetterman, G. H., Wratney, M. J., Donaldson, J. S., Danowski, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophy.+I.+History,+clinical+status,+muscle+strength,+and+biopsy+findings.">Muscular dystrophy. I. History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li><li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy.">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li><li>Mercuri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine.">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li><li>Tasca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles.">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779 (2012).</li><li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diagnostic+Reliability+of+Frozen+Sections.">The Diagnostic Reliability of Frozen Sections.</a> The American journal of pathology. 5, 377-380 (1929).</li><li>Shi, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+value+of+frozen+tissue+section+used+as+'gold+standard'+for+immunohistochemistry.">Evaluation of the value of frozen tissue section used as 'gold standard' for immunohistochemistry.</a> Am J Clin Pathol. 129, 358-366 (2008).</li><li>Turan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+frozen+sections+for+intraoperative+diagnosis+of+complex+atypical+endometrial+hyperplasia.">Accuracy of frozen sections for intraoperative diagnosis of complex atypical endometrial hyperplasia.</a> Asian Pacific journal of cancer prevention : APJCP. 13, 1953-1956 (2012).</li></ol>

The authors have nothing to disclose.

Histology and immunohistochemistry have been used as key tools in understanding the manifestation and progression of pathology in various neuromuscular disorders. Classically, muscle biopsies were first fixed in formaldehyde and then embedded with paraffin wax. While formaldehyde fixation preserves tissue architecture and allows tissue storage and transport at RT, it also inactivates enzymes and crosslinks proteins necessitating further steps to achieve accurate immunostaining. This problem is eliminated with the use of ...

Examination of muscle histology plays a critical role in the understanding of different types of neuromuscular disorders1-4. When combined with other techniques, it allows researchers to get a better idea of the manifestation and progression of pathology and can also be essential to evaluate the efficacy of a therapeutic intervention5-10. However, to be accurate, the tissues need to be preserved in the proper manner so as to preserve the physiological state immediately before excision. This requires great care during removal, preservation, sectioning, and staining.
Tissues can be prepared in two different ways for light microscopic study. The first method, formalin fixed paraffin embedded (FFPE) sections, requires tissues to be fixed in 10% natural buffered formaldehyde before being embedded with paraffin wax11,12. The fixation step helps to preserve morphology better but also cross links endogenous proteins thus necessitating intricate antigen retrieval procedures for immunostaining and eliminating the possibility of enzymatic staining on such sections12. Frozen sections, on the other hand, do not need to be pre-fixed or processed; therefore, proteins are able to retain native biological conformations13,14. Further, frozen sections also allow for quick turnaround time, which at times is necessary, for example in the intraoperative diagnosis of sarcomas and other surgical biopsies15. However, if not done properly, this method is prone to freeze damage, which introduces changes to muscle architecture making the sections unfit for histological examination. This paper will outline the most effective method to prepare muscle biopsies in order to achieve optimal staining for proper examination.

<table><tbody><tr><td>Cryostat</td><td>Leica CM 1850</td><td>Leica Microsystems Inc.</td><td>Buffalo Grove, IL 60089 U.S.A., office phone 1-800-248-0123&lt;br /&gt;Alternative: Richard Allen Scientific</td></tr><tr><td>Microscope</td><td>Nikon Eclipse 50i</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064 U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternative: Olympus</td></tr><tr><td>Image analysis software</td><td>Nikon Imaging Software Basic Research</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternatives: Image J, Metamorph</td></tr><tr><td>Isoflurane</td><td>14043-220-05</td><td>JD Medical Dist. Co., Inc.</td><td>1923 West Peoria Avenue, Phoenix, AZ 85029 U.S.A.</td></tr><tr><td>OCT</td><td>4583</td><td>Sakura Inc.</td><td>Torrence, CA 90501 U.S.A.&lt;br /&gt;Alternative source: Leica Microsystem</td></tr><tr><td>Freeze It</td><td>23-022524</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA, 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Disposable tissue mold</td><td>22-363-554</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Colorfrost plus slides</td><td>9991001</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Acetone</td><td>650501-4L</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Ethyl alcohol</td><td>HC-1300-1GL</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>2-methyl butane</td><td>M 32631-SL</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Xylene</td><td>HC-7001GA</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Cytoseal 280</td><td>8311-4</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Hematoxylin Gill #3</td><td>CS402-1D</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Eosin</td><td>6766007</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr></tbody></table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

The set-up for harvesting and freezing of tissues can be seen in Figure 1. Excised tissues should be stored on a gauze soaked in PBS to avoid drying (Figure 1A). When tissues are selected to be used for histological analysis they should be dipped in OCT and laid out on cryo mold in the proper orientation and as close to physiological length as possible to ensure accurate morphology (Figure 1B). Slurry of isopentane suitable for tissue freezing can be achieved by cooling ...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

do-s-don-ts-preparation-muscle-cryosections-for-histological

Research

JoVE Journal

Medicine

32.2K Views.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Video: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Biology

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 ...

Developmental Biology

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Neuroscience

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

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 ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

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