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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			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 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			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 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			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&#039;s and Don&#039;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 ...

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

31.5K 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

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

In Vivo saggio canina funzione muscolare

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

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Medicina

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria a Flusso test cellulare per individuare gli anticorpi per N-metil-D-aspartato Receptor o dopamina-2 recettore nel siero umano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Valutazione Ecografia base di coronaropatia Flow e flusso coronarico Reserve Utilizzando la pressione Sovraccarico modello nei topi

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Umano<em&gt; vasto laterale</em&gt; Biopsia muscolare scheletrico Utilizzando la Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissezione del Pancreas del Mouse per l'analisi istologica e Profiling metabolico

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Esame ecocardiografico ed istologico della morfologia cardiaca nel topo

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

Valutazione in vivo delle proprietà meccaniche e viscoelastiche del linguaggio del ratto

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Campionamento ed estrazione in loco di tumori cerebrali per l'analisi metabolomica e lipidomica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

Tecniche di dissezione e campionamento istologico del cuore in modelli animali di grandi dimensioni per malattie cardiovascolari

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Preparazione del tessuto miocardico umano per la coltivazione a lungo termine

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...