Encyclopedia of Experiments: Biology
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Fixação de etanol e Coloração DAPI: Um método para visualizar DNA em C. elegans
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- Comece com uma gota de solução tampão em um slide de microscópio e transfira vermes intactos para a gotícula. Uma vez que os vermes estejam no lugar, use um tecido livre de fiapos para absorver e remover o buffer. Fixar a amostra banhando os espécimes com 90% de etanol várias vezes, permitindo que ele evapore após cada aplicação.
Uma vez que o etanol tenha evaporado após a lavagem final, adicione uma solução contendo o corante nucleico ácido, DAPI, à amostra. DAPI se liga preferencialmente às bases de adenina e timina no sulco menor do DNA. Quando ligado, o complexo DAPI-DNA absorve a luz UV e emite luz azul visível.
Em seguida, adicione um conservante anti-fade para estender a vida útil da amostra. Aplique um deslizamento de tampa e sele-o no slide. Em seguida, use um microscópio fluorescente com um filtro azul para visualizar corpos DAPI, que, dependendo da célula e seu status de ciclo, podem representar cromossomos únicos, pares de cromátides irmãs ou núcleos inteiros. Neste experimento, contaremos pares de cromósias irmãs manchadas da DAPI em oócitos, para identificar cepas de tetraploides de Caenorhabditis elegans.
- As cepas tetraploides possuem 12 pares de cromossomos, que podem ser validados contando os corpos manchados de DAPI em oócitos não fertilizados. Para isso, solte de cinco a dez microlitros de tampão M9 em um slide e transfira de seis a dez tetraploides putativos para a gota. Sob um microscópio dissecador, absorva cuidadosamente a maior parte do M9 em um tecido de limpeza livre de lint.
Em seguida, deixe cair 10 microlitadores de 90% de etanol sobre os vermes e veja o etanol evaporar completamente. Assim que a evaporação terminar, repita o processo de adição de etanol e observá-lo evaporar. No total, adicione 10 microliters em quatro aplicações. Após a última gota evaporar, adicione seis microliters de DAPI a dois nanogramas por microlitra, ou uma mancha semelhante.
Para armazenamento a longo prazo dos slides, monte os worms em um anti-fade comercialmente disponível ou caseiro. Em seguida, adicione uma mancha de cobertura e sele as bordas com esmalte. Depois que o esmalte secar, os slides podem ser pontuados. Use um microscópio fluorescente e 100 x ampliação.
Primeiro, encontre o oócito não fertilizado mais maduro, que é imediatamente adjacente à espermatozoide, e ainda não entrou nem na espermatozoide ou no útero. Os corpos da DAPI aqui são presumivelmente pares de cromossomo único. Em seguida, concentre-se no núcleo do oócito e use o foco fino do microscópio para escaneá-lo lentamente, de cima para baixo, enquanto conta os corpos da DAPI. Em seguida, recontar os corpos DAPI no mesmo núcleo movendo o foco de baixo para cima.
Os oócitos do tipo selvagem têm em média seis corpos DAPI. A presença de 12 corpos DAPI indica que os animais nesta cepa são tetraploides parciais ou completos. Analise pelo menos 10 animais por cepa. Muitas vezes os pares de cromossomos estarão muito próximos ou tocando, de modo que o número de corpos DAPI é muitas vezes menor do que o número real de pares de cromossomos.
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