Questo lavoro è stato sostenuto dal National Institute of Aging (AG04418), National Institute on Drug Abuse, National Institute of Health, Karolinska Institutet Programma Graduate Partnership, Karolinska Institutet, Consiglio svedese della ricerca, Brain Power svedese, e Brain Foundation svedese. Molte grazie a Mattias Karlen e il Dr. Giuseppe Coppotelli per il supporto creativo di figura 1 e 2, rispettivamente; Karin Pernold di assistenza tecnica; e Drs. Barry J. Hoffer, Lars Olson, e Nils-Göran Larsson per molti consigli utili e di discussione.

1. Preparazione dei tessuti per criosezionamento <ol><li> Sacrificare l&#39;animale da una dislocazione cervicale o la decapitazione, secondo disponibili permettono di etica. </li><li> Raccogliere velocemente i tessuti di interesse (ad es. Cervello), e congelare rapidamente in ghiaccio secco (può richiedere il congelamento dei tessuti in isopentano o propano refrigerata con azoto liquido per ottenere morfologia ottimale). Negozio di tessuti in fogli di alluminio a -80 ° C fino al momento di sezione. </li><li> Incorpora tessuti congelati in preparazione criosezionamento. </li><li> Colleziona 14 micron sezioni al criostato a -21 ° C (potrebbe essere necessario regolare la temperatura ± 1-2 ° C). Scongelare sezioni su vetrini con blocco riscaldante, e conservare i vetrini senza coprire i vetrini a -20 ° C fino al momento dell&#39;uso. </li></ol> 2. COX istochimica <ol><li> Permettere che i vetrini ad asciugare a temperatura ambiente per 1 ora. Mettete le diapositive in una slide-colorazione da camera con carta da filtro bagnata, tagliata a strisce. Per ottenere consistenrisultati t in ogni esperimento, si raccomanda di elaborare un massimo di dieci diapositive per esperimento per minimizzare i ritardi di tempo. </li><li> Preparare sotto cappa chimica 1X DAB, 100 mM citocromo c in 0.1 M PBS pH = 7.0. Vortex rapidamente. </li><li> Aggiungere 2 mcg bovina catalasi (2 mg ml -1 o circa 4 UI ml -1). Mescolare bene nel vortex per rompere tutti i granelli di catalasi. </li><li> Applicare 150-200 ml di mezzo di incubazione di ogni diapositiva, utilizzare puntale a diffondersi in modo uniforme su tutte le sezioni. </li><li> Incubare i vetrini per 40 minuti a 37 ° C. </li><li> Rimuovere la soluzione in eccesso dalle diapositive. Lavare i vetrini 4 volte, 10 minuti ogni volta, di 0,1 M PBS pH = 7.0. </li><li> Ritorna diapositive slide-colorazione da camera con strisce di carta bagnata. </li></ol> 3. SDH istochimica <ol><li> Preparare sotto cappa chimica 1,5 mM NBT, 130 mM sodio succinato, 0,2 PMS mM e 1,0 mM di sodio azide in 0.1 M PBS pH = 7.0. Fare attenzione a proteggere ilPMS dalla luce. Vortex rapidamente. </li><li> Applicare 150-200 ml di mezzo di incubazione di ogni diapositiva, utilizzare puntale a diffondersi in modo uniforme su tutte le sezioni. </li><li> Incubare i vetrini per 40 minuti a 37 ° C. </li><li> Rimuovere la soluzione in eccesso dalle diapositive. Lavare i vetrini 4 volte, 10 minuti ogni volta, di 0,1 M PBS pH = 7.0. </li><li> Disidratano le diapositive per 2 minuti a le seguenti concentrazioni di etanolo: 70%, 70%, 95%, 95%, 99,5%. Quindi consentire 10 minuti in un ulteriore passaggio al 99,5%. </li><li> Collocare le diapositive in xilene per 10 minuti. Montare con Neo Entellan e coprioggetto. Lasciare che il vetrini si asciughino ore durante la notte, o almeno 1-2 in una zona ventilata. </li></ol> 4. Determinazione della disfunzione mitocondriale <ol><li> La quantità di disfunzione mitocondriale è indicata dal numero di cellulare colorazione blu. Per semi-quantificare tali somme, vetrini devono essere codificati e visualizzati sotto la luce-microscopia campo. Semi-quantificazione deve essere effettuata su un cieco<html> base utilizzando una scala, per esempio 0-4 (0, nessuna colorazione blu, 4, solo colorazione blu). E &#39;meglio effettuare questo tipo di semi-quantificazione in diverse sezioni di un dato soggetto / animale per calcolare un valore medio per ciascun soggetto / animale. </li><li> Le statistiche devono essere eseguiti utilizzando un test non parametrico, come Mann-Whitney o Kruskal-Wallis. </li></ol> 5. Specificità controlli appropriati <ol><li> Per i controlli di specificità per l&#39;attività COX, ripetere &quot;COX istochimica&quot; passi e aggiungere 2,5 mM di sodio azide, un inibitore terminale della catena respiratoria. </li><li> Per i controlli di specificità per l&#39;attività SDH, ripetere &quot;SDH istochimica&quot; passi con la rimozione di sodio succinato e l&#39;aggiunta di 50 malonato mM, un inibitore competitivo di SDH. </li><li> Lavare e disidratare le sezioni in una serie di etanolo, e poi montare e applicare il coprioggetto le diapositive come descritto nei punti 3,4-3,6. </li></ol> 6. Rappresentante dei risultati: tenda &quot;&gt; Lo schema complessivo della COX / SDH a ​​doppia etichettatura saggio istochimico è illustrato in Figura 2. esempi rappresentativi di appropriati COX / SDH a ​​doppia etichettatura istochimica in sezioni cerebrali da wild-type e prematuro invecchiamento topi mutatore mtDNA sono illustrate nella figura 3. La colorazione marrone scuro in topi wild-type (Figura 3, pannello di sinistra) ha mostrato la normale attività COX. cellule con difetti della catena respiratoria, indicato dalla colorazione blu, sono stati rivelati in 12 settimane di età topi mutatore mtDNA, e queste carenze si più diffusa come topi mutator mtDNA di età compresa a 46 settimane (Figura 3, il centro e il pannello di destra). Esempi di inappropriati COX / SDH a ​​doppia etichettatura in sezioni cerebrali di topi a causa di insufficiente etichettatura COX wild-type sono mostrati in figura 4. Tempo di incubazione inadeguata per la dimostrazione di attività COX, o riducendo la disponibilità di ossigeno molecolare da montavetrini la diapositiva durante l&#39;incubazione, ha provocato una deposizione ridotta del reazione DABione del prodotto e, quindi, ha permesso per la formazione del prodotto finale formazan blu durante l&#39;incubazione SDH. Attività COX e SDH possono essere indagati separatamente (Figura 5, sinistra e centro), ma l&#39;etichettatura sequenziale è utile per identificare cellule con carenze COX, a causa della formazione del precipitato blu durante l&#39;incubazione SDH (figura 3, centro e a destra). Controlli di specificità per le attività COX e SDH può essere fatto anche (Figura 5, a destra). <img alt="Figura 1" src="/files/ftp_upload/3266/3266fig1.jpg" /> Figura 1. Respiratoria mitocondriale Complessi IV. La catena respiratoria mitocondriale si trova all&#39;interno della membrana interna e comprende cinque complessi. Lo scopo della catena respiratoria è per il trasporto di elettroni dal complesso I al IV, e così facendo crea un gradiente di protoni attraverso la membrana interna utilizzata da Complesso V (ATPasi) per produrre ATP. Rosso esagoni represent subunità codificata dal mtDNA. Esagoni bianchi rappresentano subunità codificate dal DNA nucleare (si noti che Complesso II è completamente codificata dal genoma nucleare). Così, le mutazioni nel genoma mitocondriale può causare disfunzione della catena respiratoria dovuta a mutazioni nel subunità dei complessi della catena respiratoria. <img alt="Figura 2" src="/files/ftp_upload/3266/3266fig2.jpg" /> Figura 2. Diagramma di flusso del COX / SDH a doppia etichettatura saggio istochimico. Sezionare gli organi di interesse, rapidamente congelare i tessuti in ghiaccio secco, e conservarli a - 80 ° C. Raccogliere sezioni congelate e tiene a - 20 ° C fino al momento dell&#39;uso. Lasciare sezioni per asciugare a temperatura ambiente per 1 ora. Preparare il mezzo di incubazione per istochimica COX, si applicano le diapositive, e incubare per 40 minuti a 37 ° C. Lavare le sezioni in PBS 4 volte per 10 minuti ogni lavaggio. Preparare il mezzo di incubazione per istochimica SDH, si applicano alle slides, e ancora incubare per 40 minuti a 37 ° C. Lavare le sezioni di nuovo in PBS, disidratano in una serie di etanolo, e poi montare e applicare il coprioggetto le diapositive. La COX / SDH a ​​doppia etichetta sezioni sono pronti a vedere in campo chiaro microscopia entro 1-2 ore. <img alt="Figura 3" src="/files/ftp_upload/3266/3266fig3.jpg" /> Figura 3. Esempi rappresentativi di COX / SDH a doppia etichettatura. Sezioni di cervello da wild-type e prematuramente l&#39;invecchiamento nei topi mutatore mtDNA sono stati etichettati in modo sequenziale per le attività COX e SDH. (Bar Scala:. 200 micron) Attività normale COX (indicata dal colore marrone scuro) è stato mostrato in ippocampo di topi wild-type (a sinistra). Carenze COX (indicata dal colore blu) sono stati rivelati in ippocampo dal mtDNA topi mutatore (centro ea destra). C&#39;è stato un ulteriore calo dell&#39;attività COX da 46 settimane di età nel mtDNA topi mutatore, suggerendo esacerbazione diffusa di disfunzione della catena respiratoria. Il osservato mitoDisfunzione mitocondriale nel mtDNA mutator topi 12 è causato da alti livelli di mutazioni puntiformi del mtDNA come pure un aumento dei livelli di delezioni lineari 5. <img alt="Figura 4" src="/files/ftp_upload/3266/3266fig4.jpg" /> Figura 4. Esempi di inappropriati COX / SDH a doppia etichettatura. Sezioni di cervello da topi wild-type sono stati etichettati in modo sequenziale per le attività COX e SDH. (Bar Scala:. 200 micron) i tempi di incubazione inadeguata (10 e 25 minuti) per la dimostrazione di attività COX comportato una ridotta deposizione del prodotto marrone reazione DAB, rispetto ai 40 minuti di tempo di incubazione (a sinistra e al centro). I tempi di incubazione ridotto permesso per la formazione del prodotto finale formazan blu durante l&#39;incubazione SDH, fuorviante suggerire la presenza di cellule con deficienze COX. Montavetrini le diapositive durante l&#39;incubazione della COX ha portato anche nella formazione imprecise e deposizione delle reagire DABione del prodotto (a destra). <img alt="Figura 5" src="/files/ftp_upload/3266/3266fig5.jpg" /> Figura 5. Ndividual I COX SDH e di etichettatura e di controllo specificità. Sezioni di cervello da topi wild-type sono stati etichettati separatamente per le attività COX e SDH, indicato dal colore marrone scuro e il colore azzurro, rispettivamente, (a sinistra e al centro). Anche se le attività COX e SDH possono essere etichettate singolarmente, l&#39;etichettatura sequenziale si è dimostrata vantaggiosa in localizzare le cellule con disfunzione mitocondriale. Un esempio di controllo di specificità per le attività COX e SDH nel cervello da una wild-type topi ha dimostrato una mancanza di etichettatura (a destra). (Bar Scala:. 200 micron)

<ol>
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Il combinato COX / SDH metodo istochimico permette la visualizzazione di cellule con disfunzione mitocondriale. Questa tecnica, con i primi studi risalgono al 1968, resta popolare, con molti considerato il &quot;gold standard&quot; per identificare le malattie mitocondriali nei pazienti 14,19,26,27. Ora è spesso utilizzato per indagare mutazioni del mtDNA-driven invecchiamento e patologie legate all&#39;invecchiamento 12,13,18,20,21,24. La COX / SDH a doppia etichettatura metodo viene spesso utili...

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Ghiaccio secco </td><td> AGA Gas AB </td><td> blocco modulo </td><td></td></tr><tr><td> Isopentano (2-metilbutano) </td><td> Sigma-Aldrich </td><td> 277258 CAS: 78-78-4 </td><td></td></tr><tr><td> Cyrostat embedding soluzione </td><td> Sakura Finetek </td><td> Tissue Tek 4583 </td><td></td></tr><tr><td> Criostato </td><td> Microm </td><td> Microm modello HM 500M </td><td></td></tr><tr><td> Diapositive </td><td> Thermo Scientific </td><td> Super gelo Plus. Menzel Gläser J1800AMWZ </td><td></td></tr><tr><td> Coprire gli occhiali Vetro borosilicato </td><td> VWR International </td><td> 16004-098 </td><td> 24 x 50 mm </td></tr><tr><td> Filtro di carta </td><td> Munktell Filtro AB </td><td> Qualità: 1350 ArticoloNumero: 242 001 </td><td> 430 x 430 mm </td></tr><tr><td> 3,3 &#39;-diaminobenzidina tetraidrocloruro (DAB) </td><td> Sigma-Aldrich </td><td> Sigma sistema substrato liquido, D7304 </td><td></td></tr><tr><td> Citocromo c (Tipo III, dal cuore equino) </td><td> Sigma-Aldrich </td><td> C2506 CAS: 9007-43-6 </td><td></td></tr><tr><td> Bovina catalasi (dal fegato) </td><td> Sigma-Aldrich </td><td> C9322 CAS: 9001-05-2 </td><td></td></tr><tr><td> Tetrazolio (NBT) </td><td> Sigma-Aldrich </td><td> N6876 CAS: 298-83-9 </td><td></td></tr><tr><td> Sodio succinato </td><td> Sigma-Aldrich </td><td> S2378 CAS: 6106-21-4 </td><td></td></tr><tr><td> Phenazine methosulfate (PMS) </td><td> Sigma-Aldrich </td><td> P9625 CAS: 299-11-6 </td><td> PMS è sensibile alla luce. Scudo dalla luce. </td></tr><tr><td> Di sodio azide </td><td> Sigma-Aldrich </td><td> S8032 CAS: 26628-22-8 </td><td></td></tr><tr&gt; <td> Xilene </td><td> VWR International </td><td> EM-XX0060-4 </td><td></td></tr><tr><td> Entellan </td><td> VWR International </td><td> 100503-870 </td><td></td></tr><tr><td> Malonato (Acido malonico) </td><td> Sigma-Aldrich </td><td> M1296 CAS: 141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

DNA mitocondriale (mtDNA) difetti sono una causa importante di malattia e può essere alla base dell&#39;invecchiamento e all&#39;invecchiamento 1,2 alterazioni. La teoria mitocondriale dell&#39;invecchiamento suggerisce un ruolo per le mutazioni del mtDNA, che possono alterare l&#39;omeostasi bioenergetica e la funzione cellulare, nel processo di invecchiamento 3. Una ricchezza di prove è stato redatto a sostegno di questa teoria 1,4, un esempio è il mtDNA mutator mouse 5, tuttavia, il ruolo preciso dei danni del DNA mitocondriale nell&#39;invecchiamento non è del tutto compreso 6,7. Osservando l&#39;attività degli enzimi respiratori è un approccio semplice per lo studio disfunzione mitocondriale. Complesso IV o citocromo c ossidasi (COX), è essenziale per la funzione mitocondriale. La subunità catalitica del COX sono codificati da mtDNA e sono essenziali per l&#39;assemblaggio del complesso (Figura 1). Così, la sintesi corretta e la funzione sono in gran parte sulla base di mtDNA integrità 2.Anche se altri complessi respiratori potrebbero essere indagati, Complessi IV e II sono i più suscettibili di esame istochimico 8,9. Complesso II, o succinato deidrogenasi (SDH), è interamente codificato dal DNA nucleare (Figura 1), e la sua attività non è in genere affetti da compromissione del mtDNA, anche se un aumento potrebbe indicare la biogenesi mitocondriale 10-12. Il mtDNA alterato osservati in malattie mitocondriali, invecchiamento e malattie legate all&#39;età conduce spesso alla presenza di cellule con scarsa o assente attività COX 2,12-14. Anche se le attività COX e SDH possono essere analizzati individualmente, il sequenziale a doppia etichettatura 15,16 metodo ha dimostrato di essere vantaggioso per localizzare le cellule con disfunzione mitocondriale 12,17-21. Molte delle costituzioni ottimale del test sono stati determinati, come la concentrazione del substrato, accettori di elettroni / donatori, intermedio trasportatori di elettroni, l&#39;influenza del pH, e t reazioneime 9,22,23. 3,3 &#39;-diaminobenzidina (DAB) è un donatore di elettroni efficace ed affidabile 22. Nelle cellule con il funzionamento della COX, il marrone indamine polimero prodotto sarà localizzare in creste mitocondriali e saturare le cellule 22. Queste cellule disfunzionali con COX quindi non sarà saturata dal prodotto DAB, consentendo la visualizzazione di attività SDH con la riduzione del tetrazolio (NBT), accettore di elettroni, di un blu prodotto finale formazan 9,24. Citocromo c e substrati succinato di sodio vengono aggiunte per normalizzare i livelli endogeni tra controllo e malati / mutante tessuti 9. Catalasi viene aggiunto come precauzione per evitare possibili reazioni contaminazione da attività di perossidasi 9,22. Methosulfate Phenazine (PMS), un vettore intermedio di elettroni, viene utilizzato in combinazione con sodio azide, un inibitore della catena respiratoria, per aumentare la formazione di prodotti di reazione finale di 9,25. Nonostante questo informarezione, alcuni dettagli fondamentali che influenzano il risultato di questo test conveniente semplice, in aggiunta ai controlli specificità e avanzamenti nella tecnica, non sono ancora stati presentati.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Il citocromo c ossidasi / sodio deidrogenasi (COX / SDH) doppio metodo di etichettatura permette di visualizzare direttamente mitocondriale deficit enzimatici respiratori nei fresco congelato sezioni di tessuto. Questa è una tecnica semplice istochimiche ed è utile nelle indagini malattie mitocondriali, invecchiamento e patologie legate all&#39;invecchiamento.

Visualizzazione della funzione respiratoria mitocondriale utilizzando Citocromo C Ossidasi / succinato deidrogenasi (COX / SDH) Doppio etichettatura Istochimica

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

visualization-mitochondrial-respiratory-function-using-cytochrome-c

Research

JoVE Journal

Biology

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

36.5K Views.  Karolinska Institutet. Il citocromo c ossidasi / sodio deidrogenasi (COX / SDH) doppio metodo di etichettatura permette di visualizzare direttamente mitocondriale deficit enzimatici respiratori nei fresco congelato sezioni di tessuto. Questa è una tecnica semplice istochimiche ed è utile nelle indagini malattie mitocondriali, invecchiamento e patologie legate all'invecchiamento.

Video: Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Ricerca

Biologia

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

Il Ex vivo Preparazione isolato microvasi scheletrico per le Indagini di reattività vascolare

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

La valutazione delle funzioni mitocondriali e la vitalità cellulare in cellule renali proteina chinasi C overexpressing isoenzimi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualizzazione di sviluppo craniofacciale nei SOX10: Kaede transgenici Zebrafish line utilizzando Time-lapse Microscopia confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Su larga scala Knockdown Gene in<em&gt; C. elegans</em&gt; Utilizzo di librerie di alimentazione dsRNA per generare fenotipi Robusti Perdita di funzione

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Misurare funzione respiratoria nei topi mediante sfrenato Pletismografia corpo intero

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

Ad alta risoluzione respirometria mitocondriale per valutare la funzione in permeabilizzate e cellule intatte

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Studiare mitocondriale Struttura e funzione in<em&gt; Drosophila</em&gt; ovaie

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.