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 Raccolta e congelamento dei tessuti muscolari <ol><li> Euthanize il mouse con un sovradosaggio di isoflurano (2-cloro-2- (difluorometossi) -1,1,1-trifluoro-etano). Eseguire questo passaggio in una cappa aspirante e utilizzare un metodo di contenimento secondario, come un essiccatore o una campana di vetro contenente un batuffolo di cotone imbevuto di isoflurano. </li><li> Confermare la morte con fermezza spremitura zampa. Utilizzare un metodo secondario dell&#39;eutanasia come dislocazione cervicale. </li><li> Bagnare il pelo con il 70% di alcool per ridurre l&#39;incollaggio di ciocche di capelli sciolti sui muscoli. Sbucciare la pelle di dosso l&#39;arto utilizzando una pinza sottile come 5 #. </li><li> Separare cautamente il muscolo di interesse (tibiale anteriore (TA) in questo caso) dalle ossa sotto, partendo dal tendine e rimuovere. </li><li> Accise il muscolo, coprire in OCT (Optimum Cutting composto Temperature), e adagiarlo orizzontalmente su uno stampo di congelamento e getta etichettati. Assicurarsi che il muscolo è al suo normale orientamento fisiologico (testa di tail) senza stiramento. In alternativa, congelare il muscolo su piccole piazze di polistirolo e il pin con perni di insetti al fine di garantire la corretta lunghezza dei muscoli. </li><li> Raffreddare 2-metilbutano (isopentano) in un becher di acciaio con azoto liquido. Questo richiede, in media, 3-5 min. Continuate a mescolare intermittenza fino precipitati bianchi cominciano a formarsi sul fondo e sulle pareti del bicchiere. NOTA: In questo caso, isopentano ha raggiunto la temperatura ottimale di congelamento (-150 ° C) </li><li> Immergere gli stampi contenenti muscoli del refrigerata 2-metilbutano. Congelare piccoli muscoli, come il lungo del diaframma e delle dita (EDL) per 6-12 secondi e muscoli più grandi, come il soleo gastrocnemio (GS) e tricipiti per 15 - 20 sec. Non congelare i muscoli troppo a lungo come questo può portare alla rottura. </li><li> Trasferire i tessuti congelati su ghiaccio secco e lasciare evaporare isopentano (notare che questo processo richiede, in media, circa 15 - 20 min). Avvolgere i tessuti in un foglio di alluminio e memorizzarli in ultra bassa temperatura (-70 ° C a -80 ° C) fino sezionamento. </li></ol> 2. Incorporare i tessuti in ottobre preparare Tissue Block <ol><li> Tessuti di trasporto su ghiaccio secco per cyrostat per evitare lo scongelamento. Tessuto Trasferimento a criostato camera (impostato a -20 e -24 ° C per evitare la piena scongelamento dei tessuti) e lasciarli equilibrare per 30 min. </li><li> Girare la baia Peltier raffreddamento. Se il criostato non ha questo raffreddamento, utilizzare raffreddamento spray aerosol per congelare rapidamente la Ottobre Non lasciate che il disgelo muscolo in qualsiasi momento durante l&#39;incorporamento come questo può causare danni da congelamento. </li><li> Aggiungere uno strato sottile uniforme ottobre sul disco dei campioni e lasciate raffreddare. Quando la maggior parte della Ottobre è congelato sul fondo mentre ancora liquido sulla parte superiore, inserire il tessuto con l&#39;orientamento corretto (perpendicolare a ottobre per le sezioni trasversali e paralleli a ottobre per le sezioni longitudinali). Usare spruzzi di raffreddamento spray per congelare rapidamente ottobre Toccare la parte unembeded del tessuto con ext caloreRATTORE e attendere 45 secondi. </li><li> Aggiungere un altro strato sottile dell&#39;OCT intorno al muscolo, spruzzare con spray di raffreddamento aerosol ed estrarre il calore nel passaggio precedente. </li><li> Continuare ad aggiungere OCT in piccoli incrementi e rapidamente congelare la OCT utilizzando una combinazione di spray aerosol ed estrattore di calore di raffreddamento fino a quando l&#39;intero muscolo è coperto. </li><li> Mettere l&#39;estrattore di calore sulla parte superiore del blocco di tessuto e attendere per 5 min. </li></ol> 3. Preparazione Sezioni e identificare le sezioni da Mid-pancia Regione dei muscoli <ol><li> Montare il campione sulla testa dei campioni. Serrare la manopola per fissare il disco. Assicurarsi che il disco di preparato è in buon contatto con la testa del preparato. </li><li> Regolare la temperatura della camera tra il -21 e -24 ° C e attendere fino al raggiungimento della temperatura impostata. </li><li> Regolare le impostazioni di spessore (7 micron); tagliare il blocco avanzando o ritraendo il blocco utilizzando le impostazioni grossolane e fini. </li><li> Raccogliere sezioniil pre-riscaldato vetrini da microscopio carica positiva premendo la parte superiore della slitta sulle sezioni, utilizzando l&#39;attrazione tra cariche opposte per facilitare l&#39;adesione delle sezioni tagliate alla diapositiva. </li><li> Identificare sezioni metà pancia misurando sezione trasversale (CSA) delle sezioni. Fare questo software di imaging utilizzando il computer collegato al microscopio. NOTA: le immagini a contrasto di fase sono prese e la circonferenza di tutto il muscolo viene misurata tramite una funzione di traccia sul programma software. Questo produrrà sia CSA nonché diametro minimo ferret (misura della fibra dimensioni trasversali che si esprime come la distanza minima tra due tangenti paralleli sui lati opposti di una particella) misurazioni. Nota: Quando CSA e diametro minimo furetto misure cominciano a stabilizzarsi, a metà pancia è stato raggiunto. </li><li> Inventario e archiviare i vetrini a -80 ° C per il futuro colorazione. NOTA: ottobre non ha bisogno di essere rimosso prima di staininprocedure g. Una volta che le diapositive sono coprioggetto, sono pronti per essere colorati immediatamente. </li></ol> 4. Colorazione NOTA: Durante le procedure dettagliate passo-passo possono essere trovati sulla scheda tecnica del prodotto e devono essere seguite per le procedure di colorazione, ottimizzazione personale può essere richiesto per ottenere la colorazione ottimale. <ol><li> Poiché il montaggio di mezzi (vedi tabella) non è miscibile con acqua, disidratazione diapositive attraverso crescenti gradi di alcool (2 min ciascuno in 70%, 95% e 100% etanolo) e chiaro con xilene (100% per 5 min) per garantire diapositive non diventano opachi nel tempo. </li><li> Coverslip sezioni e lasciarli asciugare. Immagine con il microscopio dotato di software di imaging. </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.

Istologia e immunoistochimica sono stati utilizzati come strumenti chiave per comprendere la manifestazione e la progressione della patologia in vari disturbi neuromuscolari. Classicamente, biopsie muscolari sono stati fissati prima di formaldeide e poi integrati con paraffina. Mentre fissazione formaldeide conserva l&#39;architettura dei tessuti e permette lo stoccaggio dei tessuti e il trasporto a temperatura ambiente, è inattiva anche enzimi e proteine ​​legami crociati che richiedono ulteriori misure per raggiu...

L&#39;esame di istologia muscolare gioca un ruolo fondamentale nella comprensione dei diversi tipi di malattie neuromuscolari 1-4. Quando combinato con altre tecniche, permette ai ricercatori di avere una migliore idea della manifestazione e la progressione della patologia e possono anche essere essenziale per valutare l&#39;efficacia di un intervento terapeutico 5-10. Tuttavia, per essere precisi, i tessuti devono essere conservati in modo appropriato in modo da preservare lo stato fisiologico immediatamente prima escissione. Ciò richiede grande attenzione durante la rimozione, la conservazione, il sezionamento, e la colorazione. I tessuti possono essere preparati in due modi diversi per luce studio microscopico. Il primo metodo, fissati in formalina paraffina (FFPE) sezioni, richiede tessuti da fissare nel 10% naturale formaldeide tamponata prima di essere embedded con paraffina 11,12. La fase di fissazione aiuta a preservare la morfologia meglio, ma anche link incrociati pr endogenooteins e perciò necessitano di procedure di recupero antigene intricato per immunostaining ed eliminando la possibilità di colorazione enzimatica su tali sezioni 12. Sezioni congelate, d&#39;altra parte, non devono essere pre-fisso o trasformati; Pertanto, le proteine ​​sono in grado di trattenere conformazioni biologici nativi 13,14. Inoltre, sezioni congelate consentono anche il tempo di risposta rapido, che a volte è necessario, ad esempio nella diagnosi intraoperatoria di sarcomi e altri biopsie chirurgiche 15. Tuttavia, se non fatto correttamente, questo metodo è soggetto a danni dovuti al ghiaccio, che introduce modifiche all&#39;architettura muscolare rendendo sezioni inadatti per l&#39;esame istologico. Questo documento illustrerà il metodo più efficace per preparare biopsie muscolari per raggiungere colorazione ottimale per un esame adeguato.

<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

Valutazione istologica delle biopsie muscolari ha servito come uno strumento indispensabile per la comprensione dello sviluppo e la progressione della patologia di malattie neuromuscolari. Tuttavia, al fine di fare ciò, cura adeguata deve essere presa quando asportare e conservare tessuti per ottenere la colorazione ottimale. Un metodo di conservazione del tessuto comporta fissa tessuti in formaldeide e poi incorporare con la cera di paraffina. Questo metodo mantiene la morfologia bene e consente la memorizzazione a lungo termine a temperatura ambiente, ma è pesante e richiede una gestione di sostanze chimiche tossiche. Inoltre, i risultati di fissazione formaldeide in antigene cross-linking, che richiede protocolli di recupero antigene per immunocolorazione efficace. Al contrario, sezionamento congelati non richiede fissazione e mantiene quindi biologica dell&#39;antigene conformazione. Questo metodo fornisce anche un netto vantaggio in breve tempo di girarsi, il che rende particolarmente utile in situazioni che necessitano di una rapida valutazione istologica come intraoperativa biopsie chirurgiche. Qui si descrive il metodo più efficace di preparazione biopsie muscolari per la visualizzazione con diverse macchie istologiche ed immunologiche.

Il set-up per la raccolta e il congelamento dei tessuti può essere visto in Figura 1. Tessuti asportati devono essere memorizzati su una garza imbevuta di PBS per evitare l&#39;essiccazione (Figura 1A). Quando vengono selezionati i tessuti da utilizzare per l&#39;analisi istologica devono essere immersi in OCT e disposti su stampo crio nell&#39;orientamento corretto e più vicino alla lunghezza fisiologica possibile per garantire morfologia accurata (Figura 1B). Slurry...

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.

Cosa fare e cosa non fare nella preparazione di Muscle criosezioni per l&#39;analisi istologica

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

31.8K 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

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.

Induzione di acuta muscolo scheletrico Rigenerazione da cardiotossina Injection

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

Ricerca

Biologia dello sviluppo

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.

Preparazione della piega vocale del ratto per le analisi neuromuscolari

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

Neuroscienze

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.

Tissue Triage e Freezer per i modelli di malattia muscolare scheletrico

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

Biologia

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.

Metodo criosezionamento per Microdissezione di Murine colon 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 ...

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.

Tipo di fibra e analisi subcellulare specifica del contenuto di goccioline lipidiche nel muscolo scheletrico

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Medicina

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.

-High Throughput, Multi-Image Cryohistology di mineralizzati tessuti

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.

Criosezionamento di regioni contigue di un muscolo scheletrico mouse unico per l&#39;espressione genica e analisi istologiche

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.

Una multi-foro esageratamente Elimina gelati Imperfezioni muscoli sono direttamente immerso in azoto liquido

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

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

In Situ Immunofluorescenti colorazione di autofagia nelle cellule staminali muscolari

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

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.

Cosa fare e cosa non fare nella preparazione di Muscle criosezioni per l'analisi istologica

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