Sequential FISH Procedure

En la mayoría de los casos, independientemente de la causa, si se obtienen resultados de FISH insuficientes, se pueden volver a hibridar los portaobjetos con una nueva mezcla de sondas. Si la sonda está directamente marcada, se puede utilizar la misma u otra sonda.  

Las condiciones se basan en muestras de sangre periférica.

  1. Con unas pinzas, retire con cuidado los cubreobjetos de las dianas previamente hibridadas.
  2. Deshidrate los portaobjetos a temperatura ambiente sumergiéndolos en una serie de soluciones de lavado de etanol al 70 %, 85 % y al 100 % durante 1 minuto en cada una. Deje el portaobjetos secar al aire. Los portaobjetos están listos para repetir la hibridación. Se recomienda que el tiempo de desnaturalización de los portaobjetos se reduzca a aproximadamente el 50 % del tiempo de desnaturalización original, pero que no sea inferior a 3 minutos. Las posteriores rehibridaciones no requieren reducción adicional del tiempo de desnaturalización.

La intensidad de la tinción de contraste disminuye con las hibridaciones secuenciales, sin embargo, la pérdida es mínima. Es posible realizar hasta 8 hibridaciones repetidas de la misma metafase con diferentes sondas.

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