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1. Preparação de Sylgard cobertas de pratos, câmaras experimental, e lâminas de barbear <ol><li> 35 milímetros pratos Petri Falcon revestido com elastômero Sylgard são necessários para o corte adequado de uma retina isolada para obter células fotorreceptoras único. O elastômero é preparado de acordo com as instruções do fornecedor e uma pequena quantidade é derramado em cada prato para cobrir seu fundo com uma camada. Substitua o prato cobre após o revestimento e armazená-los. No tempo de alguns dias o elastômero endurece e os pratos estão prontos. </li><li> Fotorreceptores isolado necessidade de aderir ao fundo da câmara experimental para que sejam imobilizados durante o curso de um experimento de imagem. Isto é conseguido através do revestimento do fundo das câmaras com poli-L-lisina ou poli-L-ornitina. Adicionar 200 mL de solução 0,01% de qualquer um por câmara, e cobrir as câmaras com um papel toalha para protegê-los do pó. Depois que a solução secar, lavar as câmaras com água destilada e guarde em uma caixa fechada. Use dentro de 2 semanas. </li><li> Para limpar as câmaras no final de um experimento, lavá-los com 100% de etanol para remover todo o óleo da lente de imersão em óleo e detritos celulares. Para remover os restos celulares, use com ponta de algodão aplicadores e cuidadosamente esfregue o fundo da câmara. Depois, lave com água destilada e deixe secar antes de as câmaras de re-revestimento. </li><li> Cortar lâminas de dois gumes navalha em pedaços pequenos (8 a partir de uma lâmina) com um cortador de metal. </li></ol> 2. Preparação de soluções <ol><li> A composição das soluções depende da espécie. Para os anfíbios, o da campainha tem (em mmol / L): 110 NaCl, 2,5 KCl, 1,6 MgCl 2, 1 CaCl 2, 5 HEPES, pH = 7,55. O pH deve ser ajustado para o valor final com NaOH. Para os mamíferos, o da campainha tem (em mmol / L): 130 NaCl, KCl 5, 0,5 MgCl 2, 2 CaCl 2, 25 hemisodium-HEPES, pH = 7,40. A solução de Ringer podem ser mantidos bem fechados à temperatura ambiente por alguns meses. </li><li> Solução estoque de glicose, 1 mol / L, mantidas a -20 ° C para evitar o crescimento bacteriano. </li><li> No dia do experimento, adicione glucose para a solução de Ringer para uma concentração final de 5 mmol / L. No final do dia, descarte o da campainha que contém glicose, pois ela pode crescer bactérias. </li></ol> 3. Isolamento de retinas <ol><li> Para a excisão adequada de uma retina, é importante que o animal será de adaptação ao escuro por pelo menos 2-3 horas antes do sacrifício. Os animais devem ser adaptados à escuridão em um recipiente adequado ventilado na sala escura. </li><li> Encher dois pratos 35 milímetros Petri meio do caminho com solução de Ringer. </li><li> Sacrifício do animal sob luz vermelha ofuscante e remover os olhos. Posteriormente, todos os procedimentos são realizados sob luz infravermelha através de um microscópio de dissecação ou uma câmera com um monitor de vídeo. </li><li> Remover qualquer tecido que tenham ficado de fora da superfície do olho. Corte e retire a parte anterior, em seguida, transferir a ocular em uma das placas de Petri cheia de Ringer. </li><li> Remover o vítreo e separe cuidadosamente a retina do resto da ocular por beliscar fora ou cortar quaisquer anexos. Levante cuidadosamente a retina e separá-la completamente a partir do ocular. </li><li> Transferir a retina para a segunda placa de Petri com uma pipeta de plástico. Mantenha o prato que contém a retina em uma caixa à prova de luz. </li></ol> 4. Isolamento de células fotorreceptoras única <ol><li> Todos os procedimentos são realizados sob luz infravermelha. Corte um pedaço pequeno de retina e transferi-la com uma pipeta de plástico para um prato Sylgard cobertas. O volume da solução contendo a peça retina deve ser cerca de 250 mL. </li><li> Pegue um pequeno pedaço de lâmina de barbear com a lâmina - a borda da lâmina deve estar em cerca de 45 ° para o titular. Achatar o pedaço de retina na camada Sylgard e usando a lâmina cortar o pedaço de retina finamente, mantendo-o preso para a camada de Sylgard. </li><li> Transferir 200 mL da solução contendo as células a uma câmara experimental - deixar qualquer parte restante da retina no prato Sylgard cobertas. Mantenha a câmara com as células isoladas em uma caixa à prova de luz. </li><li> Aguarde 10 min para as células para resolver, em seguida, adicione 2-3 mL de Ringer. Nesta fase, as células isoladas podem ser carregados com um corante fluorescente especial (por exemplo Fura-2) de acordo com o protocolo de carga de corantes. </li><li> As células podem agora ser levado para o estágio do microscópio para experimentos. </li></ol> 5. Fluorescência de imagem <ol><li> Transferência da câmara para o estágio do microscópio de epifluorescência. Ajustar perfusão solução, sondas de temperatura, iluminação por infravermelhos, etc </li><li> Feche as cortinas e começar a experimentar. Ligue a luz infravermelha dentro da gaiola microscópio, foco no fundo da câmara experimental, e mover o palco à procura de células. </li></ol> 6. Resultado representantes: Fig. 1 mostra a morfologia da haste isolada saudável e cones fotorreceptores obtidas com este protocolo a partir de uma retina salamandra (Ambystoma tigrinum). Salamander células têm sido amplamente utilizadas para estudos de imagem única célula de fluorescência por causa de seu grande tamanho e sua capacidade de sobreviver por várias horas após o isolamento da retina. Além disso, a partir de uma retina salamandra pode-se obter regularmente dois haste e cones fotorreceptores. Um critério importante para a saúde das células é a presença de um elipsóide intacta (Fig. 1), a parte da célula onde as mitocôndrias estão concentrados. Quando as células são vistas sob a óptica DAPI, esta concentração de mitocôndrias dá um forte sinal de fluorescência (Fig. 2) devido à presença de NADH. Falta de um elipsóide intacta é um sinal de uma célula danificada, geralmente impróprios para experimentar. Fig. 3 mostra uma haste danificada salamandra fotorreceptoras, com um corpo celular inchado e um núcleo condensado. Tais células mostrar muito mais baixa fluorescência sob a ótica DAPI, mas visto sob a ótica FITC mostrar um sinal FAD forte na região elipsóide (provenientes de nucleotídeos de flavina oxidada e flavoproteínas). Outro critério para a saúde dos fotorreceptores isolado é sua capacidade de gerar all-trans retinol (vitamina A) em seus segmentos externos à estimulação pela luz. A geração de vitamina A requer quantidades substanciais de NADPH, que depende de uma maquinaria metabólica intacta. Figuras 4 e 5 mostram a formação de vitamina A no segmentos externos de anfíbio intacta e os bastonetes do mouse, respectivamente. <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="Figura 1"/> Figura 1. Saudável fotorreceptores única haste e cone. As células foram isoladas a partir de uma retina salamandra tigre. Fototransdução ocorre no segmento externo eo elipsóide é densamente embalado com mitocôndrias. Bastonetes são responsáveis ​​pela visão luz fraca, cones para a visão de luz brilhante. <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="Figura 2"/> Figura 2. Fluorescência de vida salamandra haste e cone. Estes são células de adaptação ao escuro, mostrando fluorescência NADH forte em seus respectivos elipsóides e não significativa de vitamina A fluorescência em seus segmentos exterior. A captura da imagem de fluorescência representa sua primeira exposição à luz visível após o período de adaptação ao escuro. <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="Figura 3"/> Figura 3. Danificado salamandra vara fotorreceptoras. O corpo da célula inchada eo núcleo condensado são indicativos de danos. A célula é oxidado e não há sinal de NADH mínima (DAPI óptica), mas muito mais forte sinal de FAD (FITC óptica). <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="Figura 4"/> Figura 4. Sapo haste com NADH e retinol. Este é um fotorreceptor da haste saudáveis ​​sapo mostrando fluorescência NADH forte na região elipsóide. Antes de exposição à luz há fluorescência mínima no segmento externo. Após a exposição à luz, há um aumento significativo na fluorescência segmento externo, devido à formação de vitamina A. <img src="/files/ftp_upload/2789/2789fig5.jpg" alt="Figura 5"/> Figura 5. Rato haste com retinol. Este é um fotorreceptor da haste saudáveis ​​rato mostrando fluorescência segmento significativo exterior após a exposição à luz, devido à formação de vitamina A. As regiões elipsóide de bastonetes mouse não mostrar um forte sinal de fluorescência.

<ol>
	<li>Burns, M. E., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Counting+Photons:+Trials+and+Trends+in+Vertebrate+Visual+Transduction.">Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction.</a> Neuron. 48, 387-401 (2005).</li><li>Ebrey, T., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+Photoreceptors.">Vertebrate Photoreceptors.</a> Prog Retin Eye Res. 20, 49-94 (2001).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+Vertebrate+Photoreceptors.">Adaptation in Vertebrate Photoreceptors.</a> Physiol Rev. 81, 117-151 (2001).</li><li>Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptor+Rhodopsin.">G Protein-Coupled Receptor Rhodopsin.</a> Annu Rev Biochem. 75, 743-767 (2006).</li><li>Saari, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+Visual+Pigment+Regeneration:+The+Friedenwald+Lecture.">Biochemistry of Visual Pigment Regeneration: The Friedenwald Lecture.</a> Invest Ophthalmol Vis Sci. 41, 337-348 (2000).</li><li>Lamb, T. D., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dark+Adaptation+and+the+Retinoid+Cycle+of+Vision.">Dark Adaptation and the Retinoid Cycle of Vision.</a> Prog Retin Eye Res. 23, 307-380 (2004).</li><li>Imanishi, Y., Lodowski, K. H., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Microscopy:+Shedding+Light+on+the+Chemistry+of+Vision.">Two-Photon Microscopy: Shedding Light on the Chemistry of Vision.</a> Biochemistry. 46, 9674-9684 (2007).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleached+Pigment+Produces+a+Maintained+Decrease+in+Outer+Segment+Ca2++in+Salamander+Rods.">Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods.</a> J Gen Physiol. 111, 53-64 (1998).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Dependent+Changes+in+Outer+Segment+Free-Ca2++Concentration+in+Salamander+Cone+Photoreceptors.">Light-Dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors.</a> J Gen Physiol. 113, 267-277 (1999).</li><li>Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Cytoplasmic+Calcium+Concentration+in+the+Rods+of+Wild-Type+and+Transducin+Knock-out+Mice.">Measurement of Cytoplasmic Calcium Concentration in the Rods of Wild-Type and Transducin Knock-out Mice.</a> J Physiol. 542, 843-854 (2002).</li><li>Leung, Y. T., Fain, G. L., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Measurement+of+Current+and+Calcium+in+the+Ultraviolet-Sensitive+Cones+of+Zebrafish.">Simultaneous Measurement of Current and Calcium in the Ultraviolet-Sensitive Cones of Zebrafish.</a> J Physiol. 579, 15-27 (2007).</li><li>Matthews, H. R., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Spot+Confocal+Technique+to+Measure+Cytoplasmic+Calcium+Concentration+in+Photoreceptors.">Laser Spot Confocal Technique to Measure Cytoplasmic Calcium Concentration in Photoreceptors.</a> Methods Enzymol. 316, 146-163 (2000).</li><li>Szikra, T., Cusato, K., Thoreson, W. B., Barabas, P., Bartoletti, T. M., Krizaj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Calcium+Stores+Regulates+Calcium+Influx+and+Signal+Transmission+in+Rod+Photoreceptors.">Depletion of Calcium Stores Regulates Calcium Influx and Signal Transmission in Rod Photoreceptors.</a> J Physiol. 586, 4859-4875 (2008).</li><li>Krizaj, D., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+Calcium+Extrusion+Mechanisms+in+the+Outer+and+Inner+Segments+of+Photoreceptors.">Compartmentalization of Calcium Extrusion Mechanisms in the Outer and Inner Segments of Photoreceptors.</a> Neuron. 21, 249-256 (1998).</li><li>Chen, C., Nakatani, K., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+Magnesium+Concentration+in+Salamander+Photoreceptor+Outer+Segments.">Free Magnesium Concentration in Salamander Photoreceptor Outer Segments.</a> J Physiol. 553, 125-135 (2003).</li><li>Chen, C., Jiang, Y., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Behavior+of+Rod+Photoreceptor+Disks.">Dynamic Behavior of Rod Photoreceptor Disks.</a> Biophys J. 83, 1403-1412 (2002).</li><li>Tsina, E., Chen, C., Koutalos, Y., Ala-Laurila, P., Tsacopoulos, M., Wiggert, B., Crouch, R. K., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+Microfluorometric+Studies+of+Reduction+and+Clearance+of+Retinal+in+Bleached+Rod+Photoreceptors.">Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors.</a> J Gen Physiol. 124, 429-443 (2004).</li><li>Ala-Laurila, P., Kolesnikov, A. V., Crouch, R. K., Tsina, E., Shukolyukov, S. A., Govardovskii, V. I., Koutalos, Y., Wiggert, B., Estevez, M. E., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle:+Dependence+of+Retinol+Production+and+Removal+on+Photoproduct+Decay+and+Cell+Morphology.">Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology.</a> J Gen Physiol. 128, 153-169 (2006).</li><li>Koutalos, Y., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluorometric+Measurement+of+the+Formation+of+All-Trans-Retinol+in+the+Outer+Segments+of+Single+Isolated+Vertebrate+Photoreceptors.">Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors.</a> Methods Mol Biol. 652, 129-147 (2010).</li><li>Wu, Q., Blakeley, L. R., Cornwall, M. C., Crouch, R. K., Wiggert, B. N., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphotoreceptor+Retinoid-Binding+Protein+Is+the+Physiologically+Relevant+Carrier+That+Removes+Retinol+from+Rod+Photoreceptor+Outer+Segments.">Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments.</a> Biochemistry. 46, 8669-8679 (2007).</li><li>Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle+and+Its+Metabolic+Support+in+Gecko+Photoreceptors.">Visual Cycle and Its Metabolic Support in Gecko Photoreceptors.</a> Vision Res. 47, 363-374 (2007).</li><li>Chen, C., Blakeley, L. R., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+All-Trans+Retinol+after+Visual+Pigment+Bleaching+in+Mouse+Photoreceptors.">Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors.</a> Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).</li></ol>

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Se saudável células isoladas não são obtidas, o problema estará, ou com o isolamento ou a saúde da retina ou com a sua corte. Normalmente, após a remoção da parte frontal do olho e do vítreo, a retina prontamente decola do epitélio pigmentar. Se não, tente retire-a a partir da periferia da ocular. Se ainda é difícil separar, uma possibilidade provável é que o animal não tenha sido de adaptação ao escuro por um período de tempo adequado, ou a luz vermelha é muito brilhante. Assegurar a adaptação es...

<table border="1"><tbody><tr><th> Nome </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Quarto escuro (100-150 pé 2) </td><td></td><td></td><td></td></tr><tr><td> Luzes vermelhas 19 </td><td></td><td></td><td> Lojas on-line </td></tr><tr><td> Fontes de luz infravermelha e visualizadores de imagens de infravermelho </td><td> FJW Optical Systems, Inc. </td><td></td><td></td></tr><tr><td> Dissecando microscópio 19 </td><td></td><td></td><td> Equipado com visores de infravermelho </td></tr><tr><td> Microscópio de epifluorescência, fechado em uma gaiola à prova de luz 19 </td><td></td><td></td><td></td></tr><tr><td> Ferramentas de dissecação (tesouras, pinças, suporte de lâmina) </td><td> Roboz ou Ferramentas Ciência Belas </td><td></td><td></td></tr><tr><td> Elastômero Sylgard </td><td> Essex (Charlotte, NC) </td><td> Sylgard kit elastômero 184 </td><td></td></tr><tr><td> Poli-L-ornitina (0,01%) </td><td> Sigma-Aldrich </td><td> P4957 </td><td></td></tr><tr><td> Poli-L-lisina (0,1%) </td><td> Sigma-Aldrich </td><td> P8920 </td><td> Diluída para 0,01% </td></tr><tr><td> Câmaras experimentais </td><td> Warner Instruments (Hamden, CT) </td><td> D3512P </td><td></td></tr><tr><td> Placas de Petri, pipetas de plástico </td><td> Fisher Scientific </td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

Na retina de vertebrados, fototransdução, a conversão da luz em sinal elétrico, é realizado pela vara e células fotorreceptoras cone 1-4. Os bastonetes são responsáveis ​​pela visão com pouca luz, cones de luz brilhante. Fototransdução ocorre no segmento externo da célula fotorreceptoras, um compartimento especializado que contém uma alta concentração de pigmento visual, o detector de luz primária. O pigmento visual é composto de um cromóforo, 11 - cis retinal, ligado a uma proteína, a opsina. Um fóton absorvida pelo pigmento visual isomeriza o cromóforo de 11 - cis para trans. Este photoisomerization traz uma mudança conformacional na pigmento visual que inicia uma cascata de reações que culmina em uma mudança no potencial de membrana, e provocar a transdução do estímulo luminoso em sinal elétrico. A recuperação da célula de estímulo de luz envolve a desativação da intermediários ativados pela luz, eo restabelecimento do potencial de membrana. Ca 2 + modula a atividade de várias enzimas envolvidas na fototransdução, e sua concentração é reduzida após estimulação luminosa. Desta forma, Ca 2 + tem um papel importante na recuperação da célula de estimulação luminosa e sua adaptação à luz de fundo. Outra parte essencial do processo de recuperação é a regeneração do pigmento visual que foi destruída durante a detecção de luz pela photoisomerization de seus 11 - cromóforo cis para trans 5-7. Esta regeneração se inicia com a libertação de todos os trans-retinal do pigmento fotoativados, deixando para trás a opsina apo-proteína. O lançado all-trans retinal é rapidamente reduzida em uma reação utilizando NADPH para all-trans retinol e opsina combina com 11 fresco - cis retinal trazidos para o segmento externo para reformar o pigmento visual. All-trans retinol é então transferido para fora do segmento externo e para as células vizinhas pela transportadora especializada Protein Retinoid Interphotoreceptor Binding (IRBP). Imagens de fluorescência de células fotorreceptoras único pode ser usado para estudar sua fisiologia e biologia celular. Ca 2 + sensíveis à corantes fluorescentes podem ser usados ​​para analisar em pormenor a interacção entre o segmento externo Ca 2 + mudanças e resposta à luz 12/08, bem como o papel de interior segmento de Ca 2 + lojas em Ca 2 + homeostase 13,14 . Corantes fluorescentes também podem ser usados ​​para medir a concentração de Mg 2 + 15, pH, e como marcadores de aquoso e compartimentos da membrana 16. Finalmente, a fluorescência intrínseca de all-trans retinol (vitamina A) pode ser usado para monitorar a cinética de sua formação e remoção das células fotorreceptoras única 17-19.

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

Um método é descrito para a preparação de células vivas único fotorreceptoras de diferentes espécies de vertebrados para imagens de fluorescência. O método pode ser usado para a imagem da fluorescência de fluoróforos endógenos, como o NADH ou vitamina A, ou que de adicionado exogenamente corantes fluorescentes sensíveis ao Ca<sup&gt; 2 +</sup&gt; Ou outros fatores.

Preparação das Condições de Vida isolaram células Fotorreceptoras Vertebrados para imagens de fluorescência

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

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12.2K visualizações.  Medical University of South Carolina. Um método é descrito para a preparação de células vivas único fotorreceptoras de diferentes espécies de vertebrados para imagens de fluorescência. O método pode ser usado para a imagem da fluorescência de fluoróforos endógenos, como o NADH ou vitamina A, ou que de adicionado exogenamente corantes fluorescentes sensíveis ao Ca 2 + Ou outros fatores.

Vídeo: 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.

Uma preparação isolada da retina para gravar Resposta Luz geneticamente identificadas células ganglionares da retina

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

Neurociência

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

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

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.

Detecção de hipóxia microrregional no Cortex Cerebral mouse por imagem de dois fótons de fluorescência endógena NADH

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.

Preparação de fatias agudas zona subventricular for Imaging Cálcio

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.

Visualização de endosome Dynamics em Viver terminais nervosos com quatro-dimensional imagens de fluorescência

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 Seccionamento Rato Retina de Preparar culturas Fotorreceptoras

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.

Imagem Ca<sup&gt; 2+</sup&gt; Dynamics nos terminais Cone fotorreceptores Axon do mouse Retina

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.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

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.

Dois métodos de descascamento para o isolamento de compartimentos celulares fotorreceptor na retina do rato para análise de proteínas

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.

Espectrometria de massa de hidrogênio/deutério-deutério para o estudo da dinâmica estrutural alfa-sinucleína sob condições fisiológicas

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