Os autores gostariam de agradecer a Matthew Florença para preparação das culturas de hipocampo. Este trabalho foi financiado por doações do Instituto Nacional dos EUA sobre Envelhecimento e Associação de Alzheimer de NZG.

1. Fixação Fixadores são cancerígenas; usar luvas e lidar com os fixadores em uma coifa. Salvo indicação em contrário, todas as incubações são realizadas em gelo e todas as soluções devem ser filtrados antes da utilização. Use microscopia eletrônica de grau de reagentes. Dia 1 <ol><li class="jove_content"> Após condições experimentais (por exemplo, injecção viral, o tratamento medicamentoso), colocar a membrana com organotípicas de fatias do hipocampo de 60 x 15 mm de poliestireno placa de Petri contendo gelada de tampão fosfato 0,1 M (tampão de Sorensen fosfato), pH 7,3. Adicionar 1 - 2 ml de tampão fosfato 0,1 M directamente em cima das fatias para mantê-las frias. </li></ol> Passo crítico: Sempre utilize buffers recém preparados e fixadores. Certifique-se de que o pH do tampão está dentro do intervalo desejado. A incapacidade de fazê-lo pode danificar estruturas celulares. <ol start="2"><li> Para isolar o subcampo CA1 do corte, usar umdescartável bisturi para cortar suavemente através da próxima fatia a DG, em paralelo com a camada de células CA1 (Figura 1). Em seguida, corte a fatia restante verticalmente para remover o subcampo CA3 eo subículo. Cortado um canto da fatia para ajudar a identificar a superfície superior do tecido. </li></ol> NOTA: No caso de nenhum de entrega viral de proteína de interesse é necessária, a fatia de tecido inteiro pode ser fixada após o meio é removido com uma suave lavagem de tampão arrefecido com gelo. Isto é então seguido por corte para fora do CA1. Passo crítico: Acompanhe a parte de cima do tecido para que corte apropriado das grades mais tarde. <ol start="3"><li> Cuidadosamente remover o tecido a partir da membrana usando a parte traseira do bisturi. Usar uma pipeta de Pasteur para transferir cuidadosamente o tecido a uma placa de 12 poços contendo 0,1 M de tampão fosfato. </li><li> Remover o tampão no poço da placa e adicionar 500 &amp; mu, l - 1 ml de fixador gelada (pH 7,3) composta do seguinte: 0,1% de ácido pícrico, paraformaldeído a 1%, e 2,5% de glutaraldeído em tampão de fosfato 0,1 M. Incubar durante 2 horas a 4 ° C. </li></ol> NOTA: ácido pícrico pode ser explosivo quando seco. Mantenha-o molhado com água em um recipiente bem fechado. Armazenar em local seco e bem ventilado. <ol start="5"><li> Remova o fixador do poço e lavar as amostras 3 vezes (20 minutos cada) em tampão de fosfato 0,1 M. </li><li> Incubar durante 40 min em 1% de ácido tânico (w / v) em 0,1 M de tampão de maleato, pH 6,0. </li><li> Lavar duas vezes (20 minutos cada) em tampão de maleato. </li><li> Incubar durante 40 min, em acetato de uranilo a 1% (w / v) em tampão de maleato no escuro. Acetato de uranilo é sensível à luz e é radioactivo. Cobrir a proveta com Parafilm ao dissolver-se e armazenar qualquer tampão não utilizado no escuro a 4 ° C. </li><li> Lavar duas vezes (20 minutos cada) em tampão de maleato. </li><li> Incubar durante 20 min em 0,5% de cloro platinaide (w / v) em tampão de maleato. </li><li> Lavar duas vezes (20 minutos cada) em tampão de maleato. Armazenar as amostras a 4 ° C até estarem prontos para processamento adicional. </li></ol> 2. Desidratação e incorporação Óxido de propileno é cancerígeno. Evite vapores por trabalhar com ele em uma coifa. Use absoluto, etanol puro a 100%, que não contém qualquer traço de água. Diluir esta etanol absoluto para produzir diferentes concentrações de desidratação. Dia 2 <ol><li> Incubar durante 5 min em etanol a 50% e, em seguida, durante 5 min em etanol a 70%. </li><li> Incubar durante 15 min, em recém-preparados a 1% de p-fenilenodiamina (PPD) em etanol a 70%. </li><li> Enxaguar três vezes em etanol a 70%. </li><li> Incubar durante 5 min em etanol a 80% e, em seguida, durante 5 min em etanol a 95%. </li><li> Incubar em etanol 100% duas vezes por 5 minutos de cada. </li><li> Prepare frascos de vidro com tampas de rosca e verifique se eles são limpos e completamente secos. Transferir as fatias de frascos de vidro erotular cada frasco simples e clara. </li><li> Incubar durante 5 min em 1:1 etanol: óxido de propileno. </li><li> Incubar em óxido de propileno a 100% duas vezes por 5 minutos de cada. </li><li> Adicionar resina (Epon) a cada frasco para dar uma mistura 1:1 com o óxido de propileno. Misture delicadamente para 2 horas. Não agite os frascos muito rigor para evitar bolhas que possam interferir com a incorporação. </li><li> Adicionar resina para fazer uma mistura 3:1 de óxido de propileno, e mistura-se durante 2 h. </li><li> Transferir a resina 100% e incubar O / N. </li></ol> Dia 3 <ol start="12"><li> Amostras sanduíche entre as tiras de plástico ACLAR e cura durante 24 horas a 60 ° C. </li></ol> 3. Corte e montagem em Grids Dia 4 <ol><li> Cortar semi-finas (0,5 mm) e as secções coradas com azul de toluidina a 1% + 1% de bórax para determinar a orientação correcta de amostras. </li><li> Corte cortes ultrafinos (60 nm) e montar em grades de níquel, uma seção por grade. </li></ol>4. Imunohistoquímica Todos os amortecedores e água deve ser filtrada antes de usar. Dia 5 <ol><li> Colocar uma gota (~ 50 ul) de tampão de Tween-20/phosphate 1% (T / PB), pH 7,5, em um pedaço de Parafilm limpo sobre uma superfície plana. Gentilmente pegar a grade com fórceps limpas pela extremidade e flutuar sobre o tampão, a secção abaixo, e incubar durante 10 min à TA. </li><li> Drenar o excesso de líquido a partir da rede, colocando-se na secção do lado de papel de filtro e, em seguida, flutuar na glicina 50 mM em T / PB, durante 15 minutos à temperatura ambiente. </li></ol> Passo crítico: Para evitar o excesso de &quot;carry-over&quot; de soluções a partir de uma gota de solução para o próximo durante as incubações, drenar o excesso de líquido por tocar suavemente a borda da grade em # 1 filtro de papel entre cada mudança de solução. Também remover qualquer solução de presa entre os braços da pinça EM prende a grade por wicking suavemente com um pedaço de papel de filtro entre os braços de pinça enquanto ensutocar os seções não seque completamente. <ol start="3"><li> Seca-se a grelha com papel de filtro e, em seguida, flutuar na solução de bloqueio contendo BSA a 2,5% e 2,5% de soro a partir do animal de que o anticorpo secundário em T / PB, durante 30 min à TA. </li><li> Incubar com o anticorpo primário (em T / PB), à TA ou O / N a 4 ° C. O tempo óptimo e a temperatura de incubação, assim como a concentração do anticorpo, devem ser determinadas experimentalmente. </li><li> Lava-se a grelha de três vezes (2 minutos cada) com T / PB. </li><li> Incubar com o anticorpo secundário (anti-coelho de 01:20 ou anti-rato acoplado a ouro de 10 nm), durante 1 h à TA. </li><li> Lavar três vezes (2 minutos cada) com T / PB. </li><li> Postfix com glutaraldeído a 2% em T / PB, durante 5 min. </li><li> Lavar três vezes (2 minutos cada) com T / PB e, em seguida, três vezes (2 minutos cada) com água filtrada. </li><li> Incubar com acetato de uranilo a 2% em água durante 10 min. </li></ol> Enquanto a grade é a coloração, a preparar uma câmara de CO 2-livre por placinga pedaço de Parafilm no centro de uma placa de Petri de vidro. Em seguida, coloca 4-6 pellets de NaOH no prato em torno do Parafilm para absorver CO 2 no ar. Manter a parte superior fechada por alguns minutos. Usar uma pipeta de Pasteur e rápida transferência de um pequeno volume de solução de citrato de chumbo de Reynold para o Parafilm no prato de Petri de vidro. Abrir a parte superior apenas o suficiente para inserir a pipeta de Pasteur para minimizar a re-introdução de CO 2 para a câmara. NOTA: Para fazer a solução de Reynold citrato de chumbo, ferver 100 ml de água deionizada em um forno de microondas e deixe esfriar em um recipiente hermético. Num balão volumétrico de 50 ml com rolha, misturar 1,33 g de nitrato de chumbo, 1,76 g de citrato de sódio e 30 ml de água fervida, mediante agitação vigorosa durante 1 min. Em seguida, agitar intermitentemente por 30 min. A esta solução turva, adicionar 8 ml de hidróxido de sódio 1 M e, lentamente, inverter o frasco algumas vezes. Solução se tornará claro. Levar a solução para 50 ml com o wa fervidater. Guardar a solução hermeticamente fechado. Se parece precipitado, descartar e fazer um novo. <ol start="11"><li> Em três copos menores preparar quente, água recém-fervida deionizada. Lavar o acetato de uranilo, por imersão a grade no primeiro béquer e agitar suavemente-la em torno de 30 seg. Repita este passo nos outros dois copos. </li><li> Abrir a parte superior da placa de Petri de vidro apenas o suficiente para colocar a grelha sobre a gota de solução de citrato de chumbo de Reynold. Se possível, use uma máscara ao fazer isto para evitar respirar em citrato de chumbo e para evitar a formação de precipitado. Incubar durante 10 min. </li><li> Abrir a parte superior apenas o suficiente para remover a rede. Evite respirar no citrato de chumbo. Lave a rede três vezes por imersão sequencialmente em três copos com água morna, recentemente fervida destilada. Deixe que a grade seco sobre um pedaço de papel de filtro, a secção para cima. A amostra está pronta para o microscópio eletrônico. </li></ol>

<ol>
	<li>Faulk, W. P., Taylor, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunocolloid+method+for+the+electron+microscope.">An immunocolloid method for the electron microscope.</a> Immunochemistry. 8, 1081-1083 (1971).</li><li>Stirling, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-+and+affinity+probes+for+electron+microscopy:+a+review+of+labeling+and+preparation+techniques.">Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques.</a> J. Histochem. Cytochem. 38, 145-157 (1990).</li><li>Phend, K. D., Weinberg, R. J., Rustioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+to+optimize+post-embedding+single+and+double+staining+for+amino+acid+neurotransmitters.">Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.</a> Journal of Histochemistry &amp; Cytochemistry. 40, 1011-1020 (1992).</li><li>Berryman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tannic+acid+on+antigenicity+and+membrane+contrast+in+ultrastructural+immunocytochemistry.">Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.</a> J. Histochem. Cytochem. 40, 845-857 (1992).</li><li>Akagi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+ultracryotomy+of+CNS+tissue+for+ultrastructural+and+immunogold+analyses.">Improved methods for ultracryotomy of CNS tissue for ultrastructural and immunogold analyses.</a> J. Neurosci. Methods. 153, 276-282 (2006).</li><li>Stirling, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructual+localization+of+lysozyme+in+human+colon+eosinophils+using+the+protein+A-gold+technique:+effects+of+processing+on+probe+distribution.">Ultrastructual localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.</a> J. Histochem. Cytochem. 37, 709-714 (1989).</li><li>Phend, K. D., Rustioni, A., Weinberg, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+osmium-free+method+of+epon+embedment+that+preserves+both+ultrastructure+and+antigenicity+for+post-embedding+immunocytochemistry.">An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.</a> Journal of Histochemistry &amp; Cytochemistry. 43, 283-292 (1995).</li><li>Zhong, L., Cherry, T., Bies, C. E., Florence, M. A., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+enhances+synaptic+strength+through+its+interaction+with+calmodulin.">Neurogranin enhances synaptic strength through its interaction with calmodulin.</a> Embo J. 28, 3027-3039 (2009).</li><li>Zhong, L., Kaleka, K. S., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+phosphorylation+fine-tunes+long-term+potentiation.">Neurogranin phosphorylation fine-tunes long-term potentiation.</a> Eur. J. Neurosci. 33, 244-250 (2011).</li><li>Zhong, L., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+targets+calmodulin+and+lowers+the+threshold+for+the+induction+of+long-term+potentiation.">Neurogranin targets calmodulin and lowers the threshold for the induction of long-term potentiation.</a> PloS one. 7, e41275 (2012).</li><li>Horisberger, M., Vauthey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelling+of+colloidal+gold+with+protein.+A+quantitative+study+using+beta-lactoglobulin.">Labelling of colloidal gold with protein. A quantitative study using beta-lactoglobulin.</a> Histochemistry. 80, 13-18 (1984).</li><li>Bendayan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+potential+and+versatility+of+colloidal+gold+cytochemical+labeling+for+molecular+morphology.">A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.</a> Biotech. Histochem. 75, 203-242 (2000).</li><li>Racz, B., Weinberg, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+coflin+in+dendritic+spines.">Spatial organization of coflin in dendritic spines.</a> J. Neuroscience. 138 (2), 447-456 (2006).</li><li>Posthuma, G., Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+the+immunogold+technique+in+quantitation+of+a+soluble+protein+in+ultra-thin+sections.">Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.</a> J. Histochem. Cytochem. 35, 405-410 (1987).</li><li>Howell, K. E., Reuter-Carlson, U., Devaney, E., Luzio, J. P., Fuller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+antigen,+one+gold?+A+quantitative+analysis+of+immunogold+labeling+of+plasma+membrane+5'-nucleotidase+in+frozen+thin+sections.">One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections.</a> Eur. J. Cell Biol. 44, 318-327 .</li><li>Yokota, E., Kawaguchi, T., Kusaba, T., Niho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Immunologic+analysis+of+families+with+complement+receptor+deficiency].">[Immunologic analysis of families with complement receptor deficiency].</a> Nihon. Rinsho. 46, 2040-2045 (1988).</li><li>Kehle, T., Herzog, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+protein-gold+complexes+and+cell+surfaces:+a+method+for+precise+quantitation.">Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation.</a> Eur. J. Cell Biol. 45, 80-87 (1987).</li><li>Bozzola, J. R., Lonnie, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Electron Microscopy. 30, (1992).</li><li>Cho, K. O., Hunt, C. A., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Hunt, C. A., Schenker, L. J., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSD-95+is+associated+with+the+postsynaptic+density+and+not+with+the+presynaptic+membrane+at+forebrain+synapses.">PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses.</a> J. Neurosci. 16, 1380-1388 (1996).</li><li>Gispen, W. H., Leunissen, J. L., Oestreicher, A. B., Verkleij, A. J., Zwiers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+localization+of+B-50+phosphoprotein:+the+(ACTH)-sensitive+protein+kinase+substrate+involved+in+rat+brain+polyphosphoinositide+metabolism.">Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.</a> Brain Research. 328, 381-385 (1985).</li><li>Biewenga, J. E., Schrama, L. H., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+phosphoprotein+B-50/GAP-43+in+neuronal+and+synaptic+plasticity.">Presynaptic phosphoprotein B-50/GAP-43 in neuronal and synaptic plasticity.</a> Acta Biochim. Pol. 43, 327-338 (1996).</li><li>Maunsbach, A. A., Bjorn, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+16.">Ch. 16.</a> Biomedical Electron Microscopy Illustrated Methods and Interpretations. , 383-426 (1999).</li></ol>

Não temos nada a revelar.

Neste protocolo, adotamos o método Phend e Weinberg para o cérebro e nos tecidos da medula espinhal para estudar espinhas dendríticas em culturas de ratos hipocampo fatia. Espinhas dendríticas na área CA3-CA1 do hipocampo são delicadas estruturas que contêm uma grande variedade de proteínas que desempenham papéis importantes na regulação de funções neuronais. O método apresentado fornece uma abordagem equilibrada para alcançar antigenicidade melhorada, mantendo boa preservação ultraestrutural (F...

<table border="1">
 <tbody>
 <tr>
 <td>NAME OF REAGENT</td>
 <td>COMPANY</td>
 <td>CATALOG NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td>60 x 15 mm polystyrene Petri dish</td>
 <td>Falcon</td>
 <td>351007</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel</td>
 <td>EXELINT</td>
 <td>29552</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture inserts</td>
 <td>Millipore</td>
 <td>PICM03050</td>
 <td></td>
 </tr>
 <tr>
 <td>10 nm Goat-anti-rabbit gold</td>
 <td>Electron Microscopy Sciences</td>
 <td>25108</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Neurogranin antibody</td>
 <td>Millipore</td>
 <td>AB5620</td>
 <td></td>
 </tr>
 <tr>
 <td>100% Picric acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>19550</td>
 <td></td>
 </tr>
 <tr>
 <td>96% Paraformaldehyde</td>
 <td>Acros Organics</td>
 <td>AC41678-0030</td>
 <td></td>
 </tr>
 <tr>
 <td>25% Glutaraldehyde (EM grade)</td>
 <td>Sigma</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Electron Microscopy Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>p-Phenylenediamine</td>
 <td>Sigma</td>
 <td>P6001</td>
 <td></td>
 </tr>
 <tr>
 <td>Platinum (IV) chloride</td>
 <td>Sigma</td>
 <td>379840</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 </tbody>
</table>

post-embedding immunogold labeling of synaptic proteins in hippocampal slice cultures

Imunomicroscopia é uma ferramenta poderosa para estudar moléculas biológicas em nível subcelular. Anticorpos acoplados a marcadores densos em electrões, tais como ouro coloidal podem revelar a localização e distribuição de antigénios específicos em diversos tecidos 1. As duas técnicas mais utilizadas são as técnicas de incorporação de pré-e pós-incorporação. Na incorporação de pré-imuno-microscopia eletrônica (ME) técnicas, o tecido deve ser permeabilizadas para permitir a penetração de anticorpos antes de ser incorporado. Estas técnicas são ideais para a preservação de estruturas, mas uma fraca penetração do anticorpo (geralmente apenas os primeiros poucos micrómetros) é um inconveniente considerável 2. Os métodos de pós-incorporação de rotulagem pode evitar este problema, porque rotulagem ocorre em seções de tecidos fixos onde os antígenos são mais facilmente acessíveis. Ao longo dos anos, um número de modificações têm melhorado os métodos de incorporação pós-para aumentar a imunorreactividade e para preservar a ultraestrutura <sup&gt; 3-5. Fixação do tecido é uma parte crucial de estudos EM. Fixadores quimicamente ligações cruzadas das macromoléculas para bloquear as estruturas de tecidos no seu lugar. A escolha de fixador afeta não só a preservação, mas também estrutural antigenicidade e contraste. Tetróxido de ósmio (OsO4), formaldeído, glutaraldeído e têm sido os fixadores padrão ao longo de décadas, incluindo para o sistema nervoso central (SNC), os tecidos que são especialmente propensas a danos estruturais durante processamento químico e físico. Infelizmente, OsO 4 é altamente reactivo e tem sido demonstrado que mascaram os antigénios 6, resultando na rotulagem pobre e insuficiente. Abordagens alternativas para evitar a fixação química incluem o congelamento dos tecidos. Mas estas técnicas são de difícil execução e exigem instrumentação dispendiosa. Para resolver alguns desses problemas e para melhorar a rotulagem SNC tecido, Phend et al. substituído OsO 4 com acetato de uranila (UA) e ACI tânicod (TA), e introduzidos com sucesso modificações adicionais para melhorar a sensibilidade da detecção de antigénio e preservação estrutural em tecidos cerebrais e da medula espinal 7. Adotamos este método ósmio livre de pós-incorporação ao tecido cerebral de ratos e otimizou a técnica de rotulagem immunogold para detectar e estudar proteínas sinápticas. Apresentamos aqui um método para determinar a localização ultra-estrutural de proteínas sinápticas em ratos hipocampais CA1 neurônios piramidais. Utilizamos fatias organotípicas de hipocampo cultivadas. Estas fatias de manter o circuito trisynaptic do hipocampo, e, portanto, são especialmente úteis para o estudo da plasticidade sináptica, um mecanismo muito pensou ser a base de aprendizagem e memória. Organotípicas de fatias de hipocampo pós-natais dias 5 e 6 de ratinho / rato pups pode ser preparado como descrito anteriormente 8, e são especialmente úteis para agudamente knockdown ou superexpressam proteínas exógenas. Nós já usou este protocolo para characterize neurogranin (Ng), uma proteína específica para neurónios, com um papel crítico na regulação da função sináptica 8,9. Temos também utilizado para caracterizar a localização ultraestrutural da calmodulina (CaM), e Ca2 + / CaM proteína-quinase dependente de II (CaMKII) 10. Como ilustrado nos resultados, este protocolo permite a preservação ultraestrutural bom das espinhas dendríticas e rotulagem eficiente de Ng para ajudar a caracterizar a sua distribuição na coluna 8. Além disso, o procedimento aqui descrito pode ter uma ampla aplicabilidade no estudo de muitas outras proteínas envolvidas em funções neuronais.

A Figura 2B mostra um exemplo de distribuição de moléculas endógenas Ng nas espinhas dendríticas de CA1 do hipocampo neurónios piramidais. Grelhas de níquel com ultrafinas (60 nm) contendo tecidos CA1 região do hipocampo (como pode ser visto na Figura 2A) foram cobertos em 1% T / PB, 50 mM de glicina, e depois bloqueadas com BSA a 2,5% e 2,5% de soro, antes da incubação com anti Ng-anticorpo. Após a lavagem com T / PB, grelhas foram então cobertas com anticorpo anti-coelho s...

Watch this Scientific Journal Video about Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures at JoVE.com

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

A localização e distribuição das proteínas fornecem informações importantes para a compreensão de suas funções celulares. A resolução espacial superior, de microscopia electrónica (ME) pode ser usado para determinar a localização subcelular de um dado antigénio imuno sequência. Para tecidos do sistema nervoso central (SNC), preservando a integridade estrutural durante a antigenicidade manutenção tem sido particularmente difícil em estudos de EM. Aqui, adoptar um procedimento que tem sido utilizada para preservar as estruturas e os antigénios do SNC para estudar e caracterizar proteínas sinápticas no hipocampo de ratos neurónios CA1 piramidais.

Pós-incorporação de Etiquetagem immunogold de proteínas sinápticas em culturas de hipocampo

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

post-embedding-immunogold-labeling-synaptic-proteins-hippocampal

Research

JoVE Journal

Neuroscience

17.0K visualizações.  Medical College of Wisconsin. A localização e distribuição das proteínas fornecem informações importantes para a compreensão de suas funções celulares. A resolução espacial superior, de microscopia electrónica (ME) pode ser usado para determinar a localização subcelular de um dado antigénio imuno sequência. Para tecidos do sistema nervoso central (SNC), preservando a integridade estrutural durante a antigenicidade manutenção tem sido particularmente difícil em estudos de EM. Aqui, ...

Vídeo: Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Preparação de Oligoméricas β-amilóide 1-42 Indução e de Impairment plasticidade sináptica em fatias do hipocampo

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Neurociência

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

O modelo de Cultura organotípicas Hippocampal Slice para o exame das lesões neuronais

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotípicas Hippocampal Culturas Slice

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

A Análise de Purkinje Morfologia de células dendríticas em Culturas fatia organotípicas

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusão lateral e Exocitose de proteínas de membrana em neurônios cultivados avaliada através da recuperação de fluorescência e fluorescência perda de fotobranqueamento

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culturas Organotípicas fatia para Estudar Oligodendrocyte Dynamics e Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Investigação de Synaptic Tagging / Captura e-captura Cruz usando aguda fatias do hipocampo de roedores

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

High-Resolution Análise Quantitativa immunogold de Receptores de Membrana na retina fita Sinapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...