Biz kalsiyum göstergelerin toplu yükleme optimize yararlı tartışmalar için Olga Garaschuk kabul etmek istiyorum.

<ol><li> Floresan kalsiyum indikatör çözeltisi hazırlayın. Biz kısa boyalar acetoxymethyl ester boyaların kullanımı, veya AM. Bunlar ekstraselüler enjekte edilen hücreler tarafından alınabilir boyalar. Biz genellikle 1 mM konsantrasyonda Oregon-Yeşil BAPTA 01:00 AM Fluo-4 kullanın. AM boyalar 50 mikrogram flakonlarda Invitrogen elde edilebilir. Biz ilk DMSO F-127 Pluronic% 20&#39;lik bir çözelti hazırlamak. Biz temizleyene kadar her gün kullanmak için önce çözüm sıcak. Biz kalsiyum göstergesi 10 mM konsantrasyon 15-20 dakika için% 20 Pluronic F-127/DMSO çözülür. 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4: Daha sonra mM içeren bir çözüm, çözüm için 1mm sulandırmak. Buna ek olarak, 100 mcM Sulforhodamine 101 genellikle etiket astrositler dahil edilir ve enjeksiyon sırasında pipet görselleştirmek. Biz 0.49 mikron santrifüj filtre (; Garaschuk ve ark, 2006 Stosiek ve ark, 2003) ile enjeksiyon için önce çözüm filtresi. </li><li> Görüntülenecek alana bir kafatası pencere İmplant. Biz <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=680" target="_blank">vallahi de genel yaklaşım tarif</a> , ancak aşağıdaki değişiklikler kalsiyum boya enjeksiyon için önemlidir: Altta yatan dura zarar vermemek için aşırı özen, ilgi kortikal alana çapı 2 mm daha küçük bir kraniotomi olun. Kraniotomi üzerinde bir yerde lamel Güvenli, ama sadece kısmen korteks oblik girmek için bir pipet için oda izin kraniotomi kenarında ve cam lamel arasında küçük bir boşluk bırakarak, kraniotomi kapsayacak. Enjeksiyonu öncesinde, kraniotomi etrafında diş çimento ile sığ bir kuyu. Iyi yönde rostral genişletmek ve enjeksiyon için mikropipet ekleme yetecek kadar sığ olmalıdır. </li><li> Mikroskop aşamasında transfer fare transferi ve bizim gösterildiği gibi, baş hareketsiz <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=678" target="_blank">kan akışını görüntülemede video</a> . </li><li> 2-4 Mega Ohm direnç veren bir pipet çektirmenin kullanarak, bir ucu çapı, cam mikroelektrot pipetler çekin. Yük, bir mikroenjektör kullanarak cam pipet üzerine kalsiyum göstergesi boya karışımı. Bir mikromanipülatör kullanarak, sonra ince bir 40X amaç altında konumlandırılmış 4X objektif kullanarak beynin yüzeyine hafifçe mikropipet düşüktür. Görüntüleme gizlemek için az ya da hiç örten kan damarları olduğu gibi enjeksiyon için uygun bir yer seçin. Iki foton mikroskobu kullanarak, pipet ucu, dura altında 200 mikrometre arasında bir derinliğe kadar görüntülenebilmekte ve korteks yavaş yavaş içine gelişmiş. Sonra, basınç, Picospritzer kullanarak 1 dakika için 10 PSI boya karışımı enjekte edilir. Biz genellikle yaklaşık 200-300 mikron uzayda ayrılmış 03:58 AM kalsiyum boya karışımı enjeksiyon, sağlar. Toplu yükleme biraz üst üste enjeksiyonları Bu yaklaşım, 600 x 600 mikrona kadar büyük bir alan, yeterince kalsiyum görüntüleme için boyanmış olmasını sağlar. Diffüz ve nöronlar tarafından alınacak boya için izin vermek için, 1 saat sonra görüntüleme AM boya karışımı enjeksiyon başlayın. </li><li> In vivo iki foton özel bir görüntüleme Chameleon Ti-Safir Lazer (800-880 nm ayarlı) ve Cambridge Teknoloji galvanometre aynalar kullanılarak yapılan iki foton mikroskop gerçekleştirin. Karel Svoboda laboratuarı (Pologruto ve ark, 2003) geliştirilen ScanImage Yazılımı kullanarak görüntü kazanır. Lazer Gücü genellikle iyi örnek az 70 mW altında. Tüm alanında görüntüler ya saniyede 1,95-15,63 kare (256 x 64 piksel 512 x 256 piksel) 20X (0.95 NA) veya 40X (0.8 NA) Olympus hedefleri kullanarak elde edilir. Hat taramaları 500 Hz elde edilir. Görüntüleme sırasında, hayvanlar, hafif izofluran anestezi altında (0.6-0.9%) tutulur ve ayrıca 37 tutulur ° C Harvard Aparatı Sıcaklık Kontrol Cihazı kullanarak. Bakım hayvanların solunum hızı 100 soluk / dk mümkün olduğunca yakın olarak tutmak için alınır. Bu seviye, anestezi kullanımı, hayvan durduran en düşük seviyede, ama yine de kayıt altına spontan aktivite izin verir. </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>

Bu yöntemin avantajı, elektrofizyolojik kayıtlar üzerinde daha az invaziv ve yoğun nöron nöron ağları faaliyet kayıtları izin vermesidir. Bu işlemi yaparken kraniotomi yaparken dura zarar vermemek için aşırı dikkat çekmek için hatırlamak önemlidir. Subdural kanama bile küçük bir miktar büyük ölçüde görüntüleme belirsiz.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

in vivo 2-photon calcium imaging in layer 2/3 of mice

Neokorteks mikrodevreler ağ dinamiklerini anlamak için, aynı anda çok sayıda nöron aktivitesini kaydetmek için esastır. In vivo iki foton kalsiyum görüntüleme, bir tek hücre çözünürlük ile yoğun bir nöronal nüfusun faaliyet kaydetmenize olanak sağlayan tek yöntem. Bu yöntem, çok sayıda nöron tarafından alınan ve son olarak in vivo iki foton mikroskop kullanarak zaman atlamalı kalsiyum görüntüleme ile nöron aktivitesini kayıt olabilir floresan kalsiyum göstergesi boya enjekte kraniyal görüntüleme penceresi implante oluşur. Astrosit özel boyalar ko-enjeksiyon astrositler gelen nöronların ayırt etmek sağlar. Bu teknik, farelerde farklı hücre grupları ağ etkileşimi daha iyi anlamak için, nöron spesifik alt popülasyonlar floresan moleküller ifade yapılabilir.

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

Neokorteks mikrodevreler ağ dinamiklerini anlamak için, aynı anda çok sayıda nöron aktivitesini kaydetmek için esastır. In vivo iki foton kalsiyum görüntüleme, bir tek hücre çözünürlük ile yoğun bir nöronal nüfusun faaliyet kaydetmenize olanak sağlayan tek yöntem.

Katman In Vivo 2-Foton Kalsiyum Görüntüleme Fare 2 / 3

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 Görüntüleme.  University of California, Los Angeles. Neokorteks mikrodevreler ağ dinamiklerini anlamak için, aynı anda çok sayıda nöron aktivitesini kaydetmek için esastır. In vivo iki foton kalsiyum görüntüleme, bir tek hücre çözünürlük ile yoğun bir nöronal nüfusun faaliyet kaydetmenize olanak sağlayan tek yöntem.

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.

Farelerde Periferik lenf nodu diseksiyonu ve 2-Foton Görüntüleme

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

Biyoloji

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.

2-Foton Mikroskop kullanarak Fare Thymus in situ Görüntüleme

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.

Kranial Pencere üzerinden neokorteks Kan Akış 2-Foton Görüntüleme Bir Yöntem

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.

Kemik İliği Üretimi 2-foton Görüntüleme Kullanım murin Dendritik Hücreler Türetilmiş

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.

Görüntüleme Protein-protein etkileşimleri 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.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

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.

FDG micro-PET/CT ile Fare Brown Fat Fonksiyonel Görüntüleme

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.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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.

Bozulmamış Arterlerin endotel katmanı Nitrik Oksit ve Süperoksit En Yüz Algılama

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.

Micropuncture tarafından 2 foton mikroskobu kolaylaştırdı farelerde Bowman'ın alanı

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