MAK riconosce il supporto per questo lavoro dai National Institutes of Health / numero - 1R15CA152985-01A1 di sovvenzione del National Cancer Institute. Questo progetto è stato supportato anche dal Miami University Undergraduate Research Award programma, le opportunità di dottorato-laurea Università di Miami per Scholarship Program e il programma di studiosi di Miami University estivo.

le procedure effettuate nel video e descritti di seguito sono state approvate da istituzionale Animal Care ed uso Committee (IACUC) presso l'Università di Miami.
 1. preparazione e Test di stimolo <ol><li>stabilire due aree distinte per la procedura chirurgica, il tavolo operatorio e la tabella post-operazione. Fase di entrambe le aree con tutti i materiali e gli utensili necessari.
	Tabella <ol><li>stage operativo sotto un cappuccio ventilato. Organizzare la tabella con l'apparecchiatura in un modo che permette il continua e senza ostacoli delle prestazioni della procedura.</li>
		<li>Stabilire una tabella di postoperatoria nella stessa stanza e vicino al tavolo principale dell'operazione. Mantenere entrambe le tabelle come ambienti sterili durante tutta la procedura.</li>
	</ol></li> <li>Posto i seguenti rifornimenti sul tavolo operatorio: un barattolo con coperchio, una provetta da 15 mL, un paio di forbici chirurgiche, una bottiglia di etanolo al 70%, due pannelli in schiuma, due pinze, due siringhe di calibro 1 mL 21, due provette da 50 mL, una provetta , un flaconcino criogenico, quattro rilievi chirurgici, dieci birilli, un dispenser di salviette sterilizzanti e un contenitore di sharps.</li>
	<li>Posto le seguenti forniture sul tavolo post-operazione: una bilancia analitica, uno 4 L dewar di azoto liquido, una vasta poco profonda bocca dewar, un rack di Conetti galleggiante, un paio di forbici chirurgiche, due pinze, quattro fiale criogeniche e un dispenser di salviette sterili.</li>
	<li>Riempire un 50 mL e un tubo da 15 mL 75% volume con formalina.</li>
	<li>Con uno spillo in ogni angolo, apporre un tampone chirurgico ad un Consiglio di schiuma approssimativo 30 x 30 cm per servire come il bordo di dissezione. Utilizzare i rimanenti quattro perni durante l'operazione. Preparare un piccolo bordo di schiuma con un tampone chirurgico per trasferire gli organi alla tabella post-op. 
	Attenzione: Isoflurano (99.9%) è una sostanza chimica tossica e deve essere utilizzato in una cappa di sfiato per garantire il massimo livello di sicurezza dall'evacuazione di gas anestetico dei rifiuti 8. Ulteriori informazioni riguardanti i rischi per i ricercatori associati all'utilizzo del metodo open-goccia con isoflurano possono essere trovate in un articolo di Taylor e Mook 8.</li>
	<li>Inserire un tampone chirurgico il vaso di vetro e bagnare con qualche goccia di isoflurane (99.9%) e posizionare un asciugamano di carta sopra la parte superiore per evitare il contatto diretto del mouse con l'isoflurano. Analogamente, utilizzare un tampone chirurgico per linea tubo restante e bagnare con qualche goccia di isoflurano e posizionare un cuscinetto supplementare sopra la parte superiore per evitare il contatto diretto tra il mouse e l'isoflurano.</li>
	<li>Pour azoto liquido in bocca ampia superficiale dewar fino alla linea di riempimento massimo è raggiunto.</li>
	<li>Posto il mouse selezionato per dissezione in anestesia da camera, cioè il vaso di vetro con un tampone imbevuto con poche gocce di isoflurane (99.9%) coperta con il coperchio, per un minimo di ~ 1 
	Nota: Questo tempo varia da mouse a mouse. Una volta che il mouse è incosciente, rimuoverlo dall'alloggiamento e posizionarlo al centro del tavolo operativo.</li>
	<li>Orientare il mouse in modo che si è sdraiato sul lato ventrale e con la testa puntato lontano dallo scienziato. Posizionare la testa all'interno del tubo interno foderato con un tampone chirurgico imbevuto con qualche goccia di isoflurane (99.9%) e un pizzico di piede per assicurare che il mouse non risponde agli stimoli per eseguire un test di stimolo.
	<ol><li>Se questo test ha esito negativo e il mouse risponde alla prova di pizzico del piede, ripetere il passaggio 1.8.</li>
	</ol></li></ol> 2. Incisione, cuore di puntura e l'eutanasia iniziale <ol><li>Pin gli arti del mouse alla scheda di schiuma chirurgica e bagnato il lato ventrale del mouse con etanolo al 70%.</li>
	<li>Pizzicare la pelle/pelliccia vicino all'apertura uretra con pinze e tirare leggermente verso l'alto. Fare un'incisione con le forbici chirurgiche attraverso la cavità addominale, a partire dall'apertura uretra, la linea mediana e termina al mento.
	<ol><li>Vicino al punto di partenza dell'incisione iniziale, afferrare un lato della pelliccia/pelle con il forcipe e fare un'altra incisione con le forbici verso il basso e in diagonale verso la zampa posteriore.</li>
		<li>Ripetere questa operazione nello stesso modo sul lato opposto. 
		Nota: La pelliccia/pelle può essere appuntata giù per creare una più ampia apertura, ma non è necessaria.</li>
	</ol></li> <li>Cuore di individuare e rimuovere il pericardio, che è la sacca intorno al cuore, per evitare l'intasamento dell'ago della siringa.
	<ol><li>Afferrare il pericardio con il forcipe e tagliare con le forbici. Eseguire la puntura di cuore inserendo con attenzione l'ago della siringa nel cuore pulsante e iniziare lentamente per ritrarre lo stantuffo.</li>
		<li>Per la raccolta del sangue ottimale, usare lo stantuffo dell'ago per imitare l'azione di pompaggio del cuore ed evitare di disegno troppo in fretta. 
		Nota: In genere circa 1 mL di sangue può essere raccolto.</li>
		<li>Dopo aver completato la raccolta di sangue, dissipare il sangue nella provetta da centrifuga e smaltire la siringa nel contenitore di sharps.</li>
		<li>Dopo la puntura di cuore viene eseguita, eseguire l'eutanasia rimuovendo gli allegati collegamento cuore. 
		Nota: L'eparina, un anticoagulante, non è stato aggiunto alla siringa in questa procedura prima della puntura cuore per permettere al sangue di coagulare per raccolta del siero in questo studio specifico. Tuttavia, se il ricercatore ha desiderato impedire la coagulazione del sangue per la raccolta del plasma, l'eparina potrebbe aggiungersi alla siringa prima della puntura di cuore.</li>
	</ol></li> <li>Se lo studio comporta la genotipizzazione del mouse, praticare una porzione dell'orecchio con le forbici e inserire in una provetta da centrifuga per una verifica di genotipo.</li></ol>
 3. pancreas estrazione <ol><li>Locate stomaco sul lato sinistro del mouse. Begin delicatamente (in modo da evitare di strappare) che separa il pancreas dallo stomaco e duodeno utilizzando due pinze. 
	Nota: Prima di scollegare il pancreas dallo stomaco e l'intestino, è molto importante che le pinze sono utilizzate delicatamente per guidare il tessuto pancreatico lontano gli organi e a non schiacciare o strappare il pancreas con il forcipe.
	<ol><li>Continuano a separare il pancreas dalle sezioni piccolo intestino digiuno e ileo e infine dall'intestino cieco dell'intestino crasso.</li>
	</ol></li> <li>In intestino cieco, riposizionare il forcipe e continuare la separazione del pancreas lungo il restante colon verso il retto. 
	Nota: A questo punto, è conveniente tagliare e rimuovere la parte dallo stomaco alla regione del colon immediatamente precedente retto.</li>
	<li>Individuare il pancreas e attaccato la milza. Far scorrere il pancreas verso il lato destro del mouse. Separare le connessioni rimanenti tra il pancreas e la cavità toracica con il forcipe per scollegare completamente il pancreas e la milza comunicanti.</li>
	<li>Rimuovere il pancreas e stenderlo per esame. Lasciare la milza associata al pancreas per scopi di identificazione.
	<ol><li>Rimuovi tutto del tessuto connettivo, il tessuto grasso e mesenterico dal pancreas. 
		Nota: Questo tessuto è più bianco nel colore e quindi può essere facilmente distinto dal tessuto del pancreas che è pinker in colore. Ciò è particolarmente importante se l'intero pancreas deve essere rimosso. Ad esempio, se il pancreas deve essere pesato e rispetto al peso corporeo o tra gruppi di animali. Nel Ptf1a cre / +; LSL-Kras G12D / + modello del topo, in particolare nei mesi precedenti, tessuto pancreatico fibroso duro potrebbe essere presente. In questo caso, la rimozione accurata del pancreas dovrà svolgersi come l'intestino potrebbe essere intrecciata nel tessuto del tumore. Nei casi avanzati, possono anche essere presenti anormale della milza e tessuto del fegato.</li>
	</ol></li> <li>Se lo si desidera, rimuovere altri organi a questo punto.</li></ol>
 4. raccolta dei dati e archiviazione <ol><li>dopo l'estrazione degli organi, spostare i campioni alla zona postoperatoria per conservazione.</li>
	<li>Pesare ogni organo e metterli nel loro rispettivo flacone criogenico. 
	Nota: lungo with la massa di ciascun campione, eventuali irregolarità devono essere registrate per riferimento futuro.</li>
	<li>Una volta che gli organi sono pesati, inserirli nell'azoto liquido per congelamento snap.</li>
	<li>Dopo lo snap di congelamento, riporre gli organi a-80 ° C per la conservazione a lungo termine.</li>
	<li>Il formalina memorizzato i campioni sulla parte superiore del banco durante la notte e la mattina successiva modificare loro soluzione da formalina in etanolo al 70%. 
	Nota: Questi campioni devono essere conservati a 4 ° C per la conservazione a lungo termine.</li>
	<li>Per immagazzinaggio a lungo termine, congelare il pugno di sangue e l'orecchio a-80 ° C.. Per la raccolta del siero dal sangue, permettere al sangue di coagulare per 30 min e poi centrifugare esso. Rimuovere la parte di siero utilizzando una pipetta e quindi conservare a -80 ° C.</li>
</ol> 5. Clean Up <ol><li>Sanitize tutti la dissezione strumenti con le salviettine sterilizzante. Tappo del tubo interno foderato con l'isoflurano imbevuto chirurgiche pad. Sostituire il tampone chirurgico sulla scheda di schiuma con un tampone chirurgico fresco. Disporre le porzioni del mouse che non sono state raccolte per l'alloggio &#39; politica di smaltimento animale s.</li>
</ol>

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Significato per quanto riguarda i metodi esistenti Mentre esista altri video informale delle dissezioni del mouse, questo video articolo fornisce la prima qualità professionale, inter pares, dimostrazione visiva di tutti i passaggi dettagliati necessari per l'estrazione e la raccolta del pancreas del mouse tramite la dissezione10 . Il pancreas è un organo principale per attività metabolica e insulino produzione, la dissezione e la raccolta del pancreas permette la conservazi...

Il mouse è emersa come un importante modello animale di cancro del pancreas umano1,2. Nel Ptf1acre / +; LSL-KrasG12D / + modello del topo, Kristen oncogene di ratto sarcoma (K-Ras) viene attivata esclusivamente nel pancreas, con conseguente apertura di lesioni precancerose del pancreas, conosciuto come neoplasia intraepiteliale pancreatica (PanINs), che il progresso per gli adenocarcinomi duttali pancreatici, comunemente come PDACs3. Questo sistema di modello del mouse fornisce uno dei migliori modelli animali disponibili per cancro del pancreas umano4,5, con l'ulteriore vantaggio che il PanINs emergono entro i primi cinque mesi di vita e frequentemente di progresso per PDAC entro un singolo anno4,5, considerando che il cancro del pancreas si verifica più frequentemente negli esseri umani 60-70 anni di età.
Estrazione del pancreas tramite la dissezione dal Ptf1acre / +; LSL-KrasG12D / + topi alle varie età permette per un esame istologico longitudinale dello sviluppo del cancro del pancreas, che vanno dalle prime fasi PanIN attraverso la progressione a PDAC3,4 , 5. raccolta del pancreas alle età che variano da cinque a quindici mesi può essere utilizzata anche per preparare il tessuto estratti per caratterizzare i cambiamenti globali nel pancreas4 metabolismo che si verificano durante la transizione da sano al tessuto malato 6,7.
Questo articolo presenta una guida visiva completa i passaggi necessari per eseguire un'estrazione del pancreas del mouse e fornisce linee guida per la conservazione di un pancreas per ulteriori analisi. Questa guida sarà altrettanto preziosa per individui conducendo una ricerca su altre malattie del pancreas, compreso il diabete di tipo e dovrebbero essere particolarmente utili per gli studenti e gli investigatori nuovi di ricerca che coinvolge la raccolta del pancreas del mouse utilizzando dissezione per profiling metabolico o analisi istologiche.

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shallow Wide Mouth Dewar</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">F3087-V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Floating Microtube Rack</td>
			<td style="width:288px;">VWR</td>
			<td style="width:119px;">60986-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryogenic Vial 1.2 mL, Sterile</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">10-500-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isothesia (Isoflurane)&#160;</td>
			<td style="width:288px;">Henry Schein Animal Health</td>
			<td style="width:119px;">050033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid Nitrogen</td>
			<td style="width:288px;">Wright Brothers</td>
			<td style="width:119px;">NIT-60-XX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse Kras Strain</td>
			<td style="width:288px;">The Jackson Laboratory</td>
			<td style="width:119px;">OO8179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse Cre Strain</td>
			<td style="width:288px;">MMRRC</td>
			<td style="width:119px;">OOO435-UNC</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of the mouse pancreas for histological analysis and metabolic profiling

Stiamo indagando il fattore di trascrizione specifico del pancreas, 1a cre-ricombinasi; ratto di salmone affumicato-fermata-salmone affumicato-Kristen sarcoma, glicina, l'acido aspartico pone al codone 12 (Ptf1acre / +; LSL-KrasG12D / +) ceppo il mouse come un modello di cancro del pancreas umano. L'obiettivo dei nostri studi attuale consiste nell'identificare nuovi biomarcatori metabolici della progressione del cancro del pancreas. Abbiamo eseguito il profilo metabolico di urina, feci, sangue ed estratti di tessuto del pancreas, come pure le analisi istologiche del pancreas per mettere in scena la progressione del cancro. Il pancreas del mouse non è un organo solido ben definito come negli esseri umani, ma piuttosto è un tessuto molle diffusamente distribuito che non è facilmente identificabile da parte di persone non hanno familiarità con l'anatomia interna del mouse o da individui che hanno poca o nessuna esperienza di prestazione dissezioni di organo del mouse. Lo scopo di questo articolo è quello di fornire una dettagliata graduale dimostrazione visiva per guidare i novizi nella rimozione del pancreas del mouse tramite la dissezione. Questo articolo dovrebbe essere particolarmente utile per gli studenti e gli investigatori di nuovo alla ricerca che richiede la raccolta del pancreas del mouse tramite la dissezione per profiling metabolico o analisi istologiche.

La figura 1 Mostra una panoramica dell'area ambiente operativa e la figura 2 Mostra l'area di operazione post. Mentre questa impostazione fornisce la quantità minima di attrezzature e messa in scena, gli individui possono scegliere di modificare questo per meglio soddisfare le esigenze individuali. Il protocollo dovrebbe essere ottimizzato secondo le specifiche esigenze dell'esperimento. Questa procedura è condotta in modo che ...

Watch this Scientific Journal Video about Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling at JoVE.com

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Questo video articolo fornisce una dimostrazione dettagliata delle procedure necessarie per rimuovere con successo il pancreas da un mouse tramite la dissezione per l'analisi istologica e profiling metabolico.

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 ...

dissection-mouse-pancreas-for-histological-analysis-metabolic

Research

JoVE Journal

Medicine

36.2K Views.  Miami University. Questo video articolo fornisce una dimostrazione dettagliata delle procedure necessarie per rimuovere con successo il pancreas da un mouse tramite la dissezione per l'analisi istologica e profiling metabolico.

Video: Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Dissezione di Human Elements corpo vitreo per l&#39;analisi proteomica

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Ricerca

Medicina

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Protocollo di raccolta per pancreas umano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Tutta vitreo Umore dissezione per l&#39;analisi Vitreodynamic

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Lesioni chirurgica al pancreas mouse attraverso la legatura del Dotto pancreatico come un modello per Endocrine e Esocrine riprogrammazione e la proliferazione

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

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.

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 ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Modello di topo per il trapianto di Pancreas usando una tecnica modificata polsino

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

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 ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

Dissezione e cultura Explant della Allantois murino per l'analisi In Vitro di allantoico allegato

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Analisi quantitativa di Micro-CT di Aortopathy in un modello murino di indotta da β-aminopropionitrile aneurisma e dissezione

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

Meccanismi di secrezione dell'ormone intestinale utilizzando l'isolato irrorati intestino tenue del ratto

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

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

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