NEI hibe EY014850 tarafından desteklenir.

1. Sylgard kapaklı sahanlar, deneysel odaları ve tıraş bıçakları hazırlanması <ol><li> Sylgard elastomer ile kaplı 35 mm Falcon Petri kutularına tek fotoreseptör hücreleri elde etmek için izole bir retina uygun kesme için gerekli. Elastomer tedarikçinin talimatlarına ve küçük bir miktar, bir tabaka ile alt kapak her çanak dökülür göre hazırlanır. Çanak kaplama sonra kapsar değiştirin ve saklayın. Elastomer birkaç gün zaman sertleşir ve bulaşıkları hazırız. </li><li> Böylece onlar bir görüntüleme deney sırasında hareketsiz İzole fotoreseptörlerin deneysel odasının alt sopa gerekir. Bu poli-L-lizin veya poli-L-ornitin odaları dipleri kaplama ile elde edilir. % 0.01 çözüm odasının her biri 200 mcL ekleyin ve tozdan korumak için bir kağıt havlu ile odaları kapsamaz. Çözüm kuruduktan sonra odaları, kapalı bir kutu distile su ve mağaza ile yıkayın. 2 hafta içinde kullanın. </li><li> Bir denemenin sonunda odaları temizlemek için herhangi bir yağ, yağ immersiyon objektif ve hücre enkaz kaldırmak için% 100 etanol ile yıkayın. Hücre enkaz kaldırmak için, pamuk uçlu aplikatörler kullanın ve odanın alt dikkatlice fırçalayın. Daha sonra, saf su ile yıkayın ve yeniden kaplama önce odaları kurumaya bırakın. </li><li> Metal kesici ile küçük parçalar (8 bir bıçak) çift kenarlı Jilet kesin. </li></ol> 2. Çözeltilerin hazırlanması <ol><li> Çözümlerinin bileşimi türüne bağlıdır. Amfibiler için, Ringer (mmol / L): 110 NaCl, 2.5 KCl, 1.6 MgCl 2, 1 CaCl 2, 5 Hepes, pH = 7.55. PH NaOH ile nihai değere ayarlanması gerekir. Memeliler için, Ringer (mmol / L): 130 NaCl, KCl 5, 0.5 MgCl 2, 2 CaCl 2, 25 hemisodium-HEPES, pH = 7.40. Ringer çözüm birkaç ay için, oda sıcaklığında iyi mühürlü muhafaza edilebilir. </li><li> Bakteri büyümesini önlemek için -20 ° C&#39;de muhafaza Stok glukoz çözeltisi, 1 mol / L. </li><li> Deney günü, son 5 mmol / L konsantrasyonu Ringer çözümü için glikoz Günün sonunda, Ringer bakterilerin üremesine çünkü glikoz içeren atın. </li></ol> 3. Retina, izolasyon <ol><li> Retinanın uygun eksizyon için hayvan kurban önce en az 2-3 saat karanlık adapte olması önemlidir. Hayvanlar karanlık oda havalandırılmış uygun bir kapta koyu adapte edilmelidir. </li><li> Yarı yolda Ringer solüsyonu ile iki adet 35 mm Petri kapları doldurun. </li><li> Loş kırmızı ışık altında hayvan kurban ve gözleri kaldırın. Daha sonra, tüm işlemleri bir diseksiyon mikroskobu veya bir video monitör ile birlikte bir kamera kullanarak kızılötesi ışığı altında yürütülmektedir. </li><li> Gözün dış yüzeyinde herhangi bir artık doku çıkarın. Kes ve ön bölümü kaldırmak, daha sonra Ringer dolu Petri kutularına biri haline Göz yuvası aktarın. </li><li> Vitreus çıkarın ve dikkatli bir kısma ya da herhangi bir ek kesme Göz yuvası geri kalanı retinanın ayrılması. Retinanın hafifçe kaldırın ve Vizör adaptör tamamen ayrı. </li><li> Ikinci Petri kabı, plastik bir transfer pipet yardımıyla retina aktarın. Retinanın bir ışık geçirmez kutu içeren çanak tutun. </li></ol> 4. Tek fotoreseptör hücrelerinin izolasyonu <ol><li> Tüm işlemler kızılötesi ışık altında yürütülmektedir. Retinanın küçük bir parça kesin ve Sylgard kaplı çanak plastik bir pipet ile transfer. Retina parça içeren solüsyon hacmi 250 mcL ilgili olmalıdır. </li><li> Jilet bıçak tutucu ile küçük bir parça Grab bıçak kenarına sahibine yaklaşık 45 ° açı olmalıdır. Sylgard tabakası retina parça düzleştirin ve Sylgard katmanı yapışmış tutarak bıçağı ince retina parça doğrayın. </li><li> Çözümün bir deney odasına hücreleri içeren 200 mcL Transferi - retinanın Sylgard kaplı bir tabak içinde kalan herhangi bir parçası bırakın. Odanın ışık geçirmez bir kutuya izole hücreleri ile tutun. </li><li> 2-3 ml Ringer eklemek, sonra yerleşmek için hücreler için 10 dakika bekleyin. Bu aşamada, boya yükleme protokole göre izole hücrelerin belirli bir floresan boya (örneğin Fura-2) ile yüklü olabilir. </li><li> Hücreler artık deneyler için mikroskop aşamasında alınabilir. </li></ol> 5. Floresans görüntüleme <ol><li> Odanın Epifloresans mikroskop sahneye aktarın. Ayarlayın çözüm perfüzyon, sıcaklık probları, kızılötesi aydınlatma, vs. </li><li> Perdeleri kapatın ve deneme başlayacaktır. Mikroskop kafes içinde kızılötesi ışık açın, deneysel odasının alt odaklanmak ve hücreler için sahne taşımak. </li></ol> 6. Temsilcisi Sonucus: Şekil 1. Bu protokol ile elde edilen bir semender (Ambystoma tigrinum) retinanın sağlıklı izole çubuk ve koni fotoreseptörlerin morfolojisi gösterir. Salamander hücreleri nedeniyle büyük boyutta ve birkaç saat sonra retina izolasyonu hayatta kalmak için yeteneklerini tek bir hücre floresan görüntüleme çalışmaları için yaygın olarak kullanılmaktadır. Buna ek olarak, bir semender retinanın bir çubuk ve koni fotoreseptörlerin hem de düzenli olarak elde edebilirsiniz. Hücrelerinin sağlığı için önemli bir kriter, sağlam bir elipsoid (Şekil 1), mitokondri yoğunlaşmıştır hücrenin bir parçası varlığıdır . Mitokondri hücreleri DAPI optik altında incelendi, bu konsantrasyon NADH varlığı nedeniyle, güçlü bir floresan sinyal (Şekil 2) verir . Sağlam bir elipsoid hasarlı hücre eksikliği genellikle deney Şekil için uygun olmayan bir işareti. 3 şişmiş bir hücre gövdesi ve yoğunlaştırılmış bir çekirdeği, bozuk bir semender çubuk fotoreseptör gösterir. Bu tür hücreler DAPI optik altında çok düşük floresan ekran, ama elipsoid bölge (okside flavin nükleotidler ve flavoproteins kaynaklanan) güçlü bir FAD sinyal göstermiyor FITC optik altında incelendi. Izole fotoreseptörlerin sağlık için bir başka kriter ışıkta stimülasyon üzerine dış segmentlerinde tüm-trans retinol (A vitamini) oluşturmak için yeteneğidir. A vitamini nesil önemli miktarda sağlam bir metabolik makine bağlıdır NADPH gerektirir. Şekil 4 ve 5 vitamini sağlam kurbağa ve fare çubuk fotoreseptör sırasıyla dış segmentlerinde bir oluşum göstermektedir. <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="Şekil 1"/> Şekil 1. Sağlıklı bir tek çubuk ve koni fotoreseptör hücrelerinin, bir kaplan semender retina izole edildi. Phototransduction dış segmentinde yer alır ve elipsoid yoğun mitokondri ile doludur. Çubuklar loş ışıkta görme, parlak ışık görüş için koniler için sorumludur. <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="Şekil 2"/> Şekil 2. Semender çubuk ve koni yaşam Floresan Bu kendi elipsoitlerin güçlü NADH floresan ve dış segmentlerinde anlamlı vitamin A floresan gösteren, koyu adapte hücrelerdir. Floresan görüntü yakalama, görünür ışık karanlık adaptasyon döneminden sonra ilk poz temsil eder. <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="Şekil 3"/> Şekil 3. Hasarlı semender çubuk fotoreseptör. Şişmiş hücre gövdesi ve özet çekirdeği hasar göstergesidir. Hücre ve okside minimal NADH sinyali (DAPI optik) var, ama çok daha güçlü FAD sinyali (FITC optik). <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="Şekil 4"/> Şekil 4. NADH ve retinol ile Kurbağa çubuk elipsoid bölgede güçlü NADH floresan gösteren sağlıklı bir kurbağa çubuk fotoreseptör. Önce dış segmente minimal floresan ışığa maruz kalma var. Işığa maruz kalma ardından, A vitamini oluşumu nedeniyle, dış segmentinde floresan önemli bir artış var. <img src="/files/ftp_upload/2789/2789fig5.jpg" alt="Şekil 5"/> Şekil 5. Retinol ile Fare çubuk Bu sağlıklı bir fare fare çubuk fotoreseptörlerin elipsoid bölgelerinde güçlü bir floresan sinyal görünmüyor A vitamini oluşumu nedeniyle ışık maruz kaldıktan sonra önemli dış segmente floresan gösteren çubuk fotoreseptör.

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Izole sağlıklı hücreler elde değilseniz, retinanın izolasyonu veya sağlık sorunu ya da doğrama ya yatıyor. Tipik olarak, göz ve vitreus ön çıkardıktan sonra, retina pigment epitel kolayca havalandığı. Göz yuvası çevreden başlayarak kapalı değilse, bunu kabuğu deneyin. Hala ayırmak için zor ise, daha büyük bir olasılık hayvan yeterli bir zaman dilimi için koyu adapte olmamıştır, ya da kırmızı ışık çok parlak. Hayvan için uygun karanlık adaptasyonu, ve loş kırmızı ışık ol...

<table border="1"><tbody><tr><th> Isim </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Karanlık oda (100-150 ft 2) </td><td></td><td></td><td></td></tr><tr><td> Kırmızı ışık 19 </td><td></td><td></td><td> Online mağazalar </td></tr><tr><td> Kızılötesi ışık kaynakları ve kızılötesi görüntü izleyiciler </td><td> FJW Optik Systems, Inc. </td><td></td><td></td></tr><tr><td> Mikroskop 19 Diseksiyon </td><td></td><td></td><td> Infrared izleyiciler ile donatılmış </td></tr><tr><td> Epifloresans mikroskop ışık geçirmeyen bir kafesin 19 içine </td><td></td><td></td><td></td></tr><tr><td> Diseksiyon araçları (makas, forseps, bıçak tutucu) </td><td> Roboz veya Güzel Bilim Araçları </td><td></td><td></td></tr><tr><td> Sylgard elastomer </td><td> İstanbul (Charlotte, NC) </td><td> Sylgard 184 elastomer kiti </td><td></td></tr><tr><td> Poli-L-ornitin (% 0.01) </td><td> Sigma-Aldrich </td><td> P4957 </td><td></td></tr><tr><td> Poli-L-lisin (% 0.1) </td><td> Sigma-Aldrich </td><td> P8920 </td><td> % 0.01 ile seyreltilir </td></tr><tr><td> Deneysel odaları </td><td> Warner Instruments (Hamden, CT) </td><td> D3512P </td><td></td></tr><tr><td> Petri kapları, plastik pipetler </td><td> Fisher Scientific </td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

Gözdeki, phototransduction ışık, bir elektrik sinyali dönüşüm, çubuk ve koni fotoreseptör hücrelerinin 1-4 tarafından yürütülmektedir. Rod fotoreseptörlerin loş ışıkta görme, parlak ışık konileri sorumludur. Phototransduction fotoreseptör hücre, görsel pigment, birincil ışık detektörü yüksek konsantrasyonda içeren özel bir bölmesi dış segmentinde yer alır. Retina cis, protein, opsin bağlı bir kromofor, 11 görme pigmenti oluşur. Görsel pigment tarafından emilir bir foton 11 kromofor isomerizes - cis-trans. Bu photoisomerization membran potansiyeli bir değişiklik sonuçlanan ve bir elektrik sinyali ışık uyaran transdüksiyon hakkında getirerek reaksiyonlar zincirini başlatan görsel pigment konformasyonel değişikliği beraberinde getiriyor. Işık uyarımı hücre kurtarma ışık ile aktive ara devre dışı bırakma ve membran potansiyeli yeniden kurulması içerir. Ca 2 + birkaç phototransduction yer alan enzimlerin aktivitesini modüle ve konsantrasyonu ışık uyarımı üzerine azalır. Bu şekilde, Ca 2 + hücre arka plan ışığı, ışık uyarılması ve adaptasyonu kurtarma önemli bir rol oynar . All-trans 5-7 cis kromofor iyileşme sürecinin bir diğer önemli bölümü, 11 photoisomerization ışık algılama sırasında tahrip olmuştur görsel pigment rejenerasyon. Bu yenilenme, apo-protein opsin geride bırakarak, all-trans retina photoactivated pigmenti tarafından serbest bırakılması ile başlar . Serbest bırakılan all-trans retina all-trans retinol, ve opsin NADPH kullanan bir tepki olarak hızla azalır, taze 11 ile birleşir - cis retina, görme pigmenti reform dış segmente getirilen. All-trans retinol sonra uzman taşıyıcı Interphotoreceptor retinoid Bağlayıcı Protein (IRBP) dış segmente ve komşu hücre içine transfer edilir. Floresan görüntüleme tek fotoreseptör hücrelerinin fizyolojisi ve hücre biyolojisi okumak için kullanılabilir. Ca 2 + duyarlı floresan boyalar 8-12 iç segmenti Ca 2 Ca 2 + mağazalar rolünün yanı sıra ışık dış segmente Ca 2 + değişiklikleri ve tepki arasındaki etkileşimi ayrıntılı olarak incelemek için kullanılabilir + homeostazı 13,14 . Floresan boyalar Mg 2 ölçmek için kullanılabilir + konsantrasyonu 15, pH, sulu ve membran bölmeleri 16 izleyiciler olarak. Son olarak, tüm-trans retinol (A vitamini) içsel floresan 17-19 tek fotoreseptör hücrelerinin oluşumu ve kaldırma kinetiği izlemek için kullanılabilir.

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

Floresan görüntüleme için farklı omurgalı türlerin tek yaşam fotoreseptör hücrelerinin hazırlanması için bir yöntem açıklanmıştır. Yöntem, NADH ya da A vitamini gibi endojen fluorophores, floresan görüntü veya Ca duyarlı Eksojen olarak eklenmiş floresan boyalar olabilir<sup&gt; 2 +</sup&gt; Ya da diğer faktörler.

Floresans Görüntüleme Living İzole Omurgalı fotoreseptör hücrelerinin hazırlanması

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

preparation-living-isolated-vertebrate-photoreceptor-cells-for

Research

JoVE Journal

Neuroscience

12.2K Görüntüleme.  Medical University of South Carolina. Floresan görüntüleme için farklı omurgalı türlerin tek yaşam fotoreseptör hücrelerinin hazırlanması için bir yöntem açıklanmıştır. Yöntem, NADH ya da A vitamini gibi endojen fluorophores, floresan görüntü veya Ca duyarlı Eksojen olarak eklenmiş floresan boyalar olabilir 2 + Ya da diğer faktörler.

Video: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Genetik Etiketli Retina Ganglion Hücreleri Işık Tepkisi Record İzole Retina Hazırlık

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Nörobilim

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Endojen NADH Floresans iki-foton Imaging tarafından Fare Serebral Korteks olarak Microregional Hipoksi Algılama

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Kalsiyum Görüntüleme için akut subventricular dilimi Dilimleri hazırlanması

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Dört boyutlu Floresans Görüntüleme ile Sinir Terminalleri Yaşayan endozom Dinamiği Görselleştirme

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Vibratome Kesit Fare Retina fotoreseptör Kültürler hazırlayın

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Görüntüleme Ca<sup&gt; 2+</supFare Retina Koni fotoreseptör Akson Terminalleri içinde&gt; Dinamikleri

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Yüksek içerik miktar Sod1 canlı hücreler Mutant Protein toplama oluşumu için tahlil geliştirme

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Protein Analizi için Fare Retinasındaki Fotoreseptör Hücre Bölmelerinin İzolasyonu İçin İki Peeling Yöntemi

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Fizyolojik Koşullar Altında Alfa-Sinüklein Yapısal Dinamiğinin İncelenmesi için Milisaniye Hidrojen / Döteryum Değişim Kütle Spektrometresi

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...