Vorremmo ringraziare Olga Garaschuk per le discussioni utili su come ottimizzare il caricamento di massa degli indicatori di calcio.

<ol><li> Preparare la soluzione di indicatore fluorescente calcio. Noi usiamo coloranti estere acetossimetil o AM coloranti in breve. Si tratta di coloranti che possono essere captata dalle cellule quando iniettate extracellulare. Usiamo tipicamente Oregon-Green Bapta-1-AM o Fluo 04:00 ad una concentrazione di 1 mM. Coloranti AM può essere ottenuto da Invitrogen in flaconcini da 50 microgrammi. Per prima cosa preparare una soluzione al 20% del Pluronic F-127 in DMSO. Noi caldo la soluzione prima dell&#39;uso tutti i giorni fino al suo chiaro. Noi sciogliere l&#39;indicatore di calcio del F-127/DMSO Pluronic 20% per 15-20 minuti ad una concentrazione di 10 mm. Abbiamo poi diluire la soluzione a 1 mm in una soluzione contenente, in mm: 150 NaCl, 2.5 KCl, 10 Hepes, pH 7,4. Inoltre, 100 mM solforodamina 101 è solitamente incluso per astrociti etichetta e visualizzare la pipetta durante l&#39;iniezione. Noi filtrare la soluzione prima di iniezione con un filtro di 0,49 micron centrifuga (Stosiek et al, 2003;.. Garaschuk et al, 2006). </li><li> Impiantare una finestra cranica sopra l&#39;area da acquisire. Abbiamo <a href="http://jove.com/index/details.stp?ID=680" target="_blank">descritto l&#39;approccio generale in JOVE</a> , ma le seguenti modifiche sono importanti per l&#39;iniezione di calcio colorante: Fai una craniotomia minore di 2 mm di diametro su tutta l&#39;area corticale di interesse, facendo attenzione estrema a non danneggiare la sottostante dura. Fissare un coprioggetto in posizione sopra la craniotomia, ma solo parzialmente coprire la craniotomia, lasciando un piccolo spazio tra il bordo della craniotomia e il coprioggetto di vetro per lasciare spazio per una pipetta per entrare nella corteccia obliquamente. Prima dell&#39;iniezione, fare un pozzo poco profondo con cemento dentale in tutto il craniotomia. Il bene dovrebbe estendono in direzione rostrale ed essere poco profondo abbastanza da consentire l&#39;inserimento della micropipetta per l&#39;iniezione. </li><li> Trasferire il mouse trasferite alla fase del microscopio e immobilizzare la testa, come mostrato nel nostro <a href="http://jove.com/index/details.stp?ID=678" target="_blank">video di imaging del flusso sanguigno</a> . </li><li> Tirare pipette microelettrodo vetro a un diametro punta cedendo 2-4 Mega Ohm della resistenza, con un estrattore pipetta. Caricare l&#39;indicatore mix di calcio inchiostri a pigmenti sul pipetta di vetro con una microsiringa. Utilizzando un micromanipolatore, abbassare delicatamente la micropipetta sulla superficie del cervello con un obiettivo 4X, che viene poi finemente posizionato sotto un obiettivo 40X. Scegliere un luogo adatto per l&#39;iniezione, in modo tale che ci sono pochi o nessun vasi sanguigni sovrastante per oscurare l&#39;imaging. Utilizzando la microscopia a due fotoni, la punta della pipetta è visualizzato e avanzava lentamente nella corteccia, ad una profondità di 200 micrometri al di sotto della dura. Poi, la pressione iniettare la miscela colorante a 10 PSI per 1 minuto usando un Picospritzer. Noi di solito consegnare 2-4 iniezioni di miscela colorante calcio AM, separati nello spazio da circa 200-300 micron. Questo approccio caricamento di massa di iniezioni leggermente sovrapposte assicura che un&#39;area grande come 600 x 600 micron è adeguatamente macchiato per l&#39;imaging di calcio. Iniziare l&#39;imaging 1 ora dopo l&#39;iniezione della miscela AM colorante, per consentire la tintura di diffondere e di essere ripreso da neuroni. </li><li> Eseguire in vivo a due fotoni di imaging con una misura microscopio a due fotoni con un Ti-Sapphire Chameleon Laser (sintonizzati su 800-880 nm) e di Cambridge specchi galvanometro Technology. Noi acquisire immagini con il software scanimage sviluppato nel laboratorio di Karel Svoboda (Pologruto et al., 2003). Potenza del laser è in genere ben inferiore a 70 mW a campione. Immagini intero campo vengono acquisiti sia mediante un 20X (0,95 NA) o 40X (0,8 NA) obiettivi Olympus, a 1,95-15,63 fotogrammi al secondo (512 x 256 pixel a 256 x 64 pixel). Scansioni linea vengono acquisiti a 500 Hz. Durante l&#39;imaging, gli animali sono tenuti sotto la luce anestesia isoflurano (0,6-0,9%), e sono anche tenuti a 37 ° C utilizzando un dispositivo di controllo della temperatura di Harvard Apparatus. Si ha cura di tenere il ritmo respiratorio di animali più vicino a 100 respiri / minuto possibile. Questo livello di utilizzo dell&#39;anestesia è il più basso livello che immobilizza l&#39;animale, ma permette comunque l&#39;attività spontanea da registrare. </li></ol>

<ol>
	<li>Garaschuk, O., Milos, R. I., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+bulk-loading+of+fluorescent+indicators+for+two-photon+brain+imaging+in+vivo.">Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.</a> Nat Protoc. 1, (2006).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+two-photon+calcium+imaging+of+neuronal+networks.">In-vivo two-photon calcium imaging of neuronal networks.</a> Proc Natl Acad Sci USA. 100, 7319-7324 (2003).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.&amp;nbsp;&amp;nbsp;+Biomed+Eng+Online+2:13.">ScanImage: flexible software for operating laser scanning microscopes.&amp;nbsp;&amp;nbsp; Biomed Eng Online 2:13.</a> Biomed Eng Online. 2, (2003).</li></ol>

Il vantaggio di questo metodo rispetto registrazioni elettrofisiologiche è che è meno invasivo e permette le registrazioni di attività nel fitto reti neuronali neuronali. Nel fare questa procedura s importante ricordarsi di prendere la massima cura quando si esegue la craniotomia non danneggiare la dura. Anche una piccola quantità di sanguinamento subdurale con molto oscuro l&#39;imaging.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Oregon Green BAPTA1-AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>O-6807</td>
<td>&nbsp;</td>
<tr>
<td>Fluo 4- AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14201</td>
<td>&nbsp;</td>
<tr>
<td>Sulforhodamine 101</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>S-359</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic F-127 in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Centrifugal Filter (0.45 micron)</td>
<td>Reagent</td>
<td>Millipore</td>
<td>UFC3 0HV 0S</td>
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in vivo 2-photon calcium imaging in layer 2/3 of mice

Per capire le dinamiche della rete di microcircuiti nella neocorteccia, è essenziale per registrare contemporaneamente l&#39;attività di un gran numero di neuroni. In vivo a due fotoni di imaging del calcio è l&#39;unico metodo che permette di registrare l&#39;attività di una densa popolazione neuronale con cella singola risoluzione. Il metodo consiste nel impiantare una finestra del cranio di imaging, l&#39;iniezione di un colorante fluorescente indicatore di calcio che può essere occupato da un gran numero di neuroni e, infine, registrare l&#39;attività dei neuroni, con immagini di calcio lasso di tempo utilizzando un in-vivo due microscopio fotone. Co-iniezione di astrociti specifici coloranti permette di differenziare i neuroni da astrociti. La tecnica può essere eseguita in topi che esprimono molecole fluorescenti in specifiche sottopopolazioni di neuroni per comprendere meglio le interazioni di rete dei vari gruppi di cellule.

Watch this Scientific Journal Video about In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice at JoVE.com

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Per capire le dinamiche della rete di microcircuiti nella neocorteccia, è essenziale per registrare contemporaneamente l&#39;attività di un gran numero di neuroni. In vivo a due fotoni di imaging del calcio è l&#39;unico metodo che permette di registrare l&#39;attività di una densa popolazione neuronale con cella singola risoluzione.

In Vivo 2-Photon Imaging di calcio a livello 2 / 3 dei topi

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

in-vivo-2-photon-calcium-imaging-in-layer-23-of-mice

Research

JoVE Journal

Biology

28.1K Views.  University of California, Los Angeles. Per capire le dinamiche della rete di microcircuiti nella neocorteccia, è essenziale per registrare contemporaneamente l'attività di un gran numero di neuroni. In vivo a due fotoni di imaging del calcio è l'unico metodo che permette di registrare l'attività di una densa popolazione neuronale con cella singola risoluzione.

Video: In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissezione e 2-Photon Imaging di linfonodi periferici nei topi

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Ricerca

Biologia

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Imaging in situ del Timo mouse utilizzando 2-Photon Microscopia

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

Un metodo per 2-Photon Imaging del flusso sanguigno nella neocorteccia attraverso una finestra del cranio

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Generazione di cellule derivate dal midollo osseo dendritiche murini per l&#39;uso in 2-fotone Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging interazioni proteina-proteina In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imaging Forma Modifica cella in Living Drosophila Embrioni

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imaging funzionale del grasso bruno nei topi con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Face Detection di ossido nitrico e superossido in strato endoteliale delle arterie Intact

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Micropuntura dello spazio di Bowman in topi nei paraggi 2 Photon microscopia

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...