Encyclopedia of Experiments: Biology
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Tinción roja del nilo de C. elegans: Un método para visualizar las gotas lipídicas en animales fijos
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- Al visualizar gotas lipídicas, los orgánulos de almacenamiento intracelular para lípidos neutros, comienzan añadiendo una solución detergente, como una que contiene Tritón X-100, a la muestra.
Luego, añade Nilo Rojo a la muestra y protégelo de la luz, ya que el tinte es sensible a la luz.
Cuando se expone a lípidos polares, como los fosfolípidos, el espectro de emisiones de Nile Red está en las longitudes de onda rojas más altas. Cuando se expone a lípidos neutros, como triacylglicerols y ésteres de esterol en el núcleo neutro de las gotas lipídicas, el espectro de emisión de Nile Red cambia a las longitudes de onda inferiores de oro amarillo.
Por lo tanto, utilice el canal verde de un microscopio fluorescente, que recogerá las emisiones de longitud de onda amarillo-oro, para visualizar la tinción roja del Nilo de gotas lipídicas en todo el animal.
- Llevar a cabo la tinción lipídica roja del Nilo, chapar gusanos en el medio de crecimiento de nematodos, o NGM, sembrar con el tronco tardío OP50 E. coli, y hacer crecer los gusanos a 20 grados Celsius a principios de la etapa L4. Lave los gusanos con 1 mililitro de solución PBST y transfiera la suspensión del gusano a un tubo de microfugio de 1,5 mililitros.
Centrífuga los gusanos a 560 veces la gravedad durante un minuto, luego retire el sobrenadante y repita el lavado PBST hasta que el E. coli sea despejado de la suspensión.
- La adición de isopropanol es crucial para la permeabilización adecuada de los gusanos antes de mancharse.
- Centrífuga los gusanos a 560 veces la gravedad durante un minuto y retire el sobrenadante sin interrumpir el pellet de gusano.
- Es importante minimizar la exposición a la luz durante los pasos de centrifugación.
- Ahora, mientras está en la oscuridad, añadir 600 microlitros de solución de trabajo NR previamente preparada a cada muestra. Invertir los tubos tres veces y mezclar completamente los gusanos y la solución.
Después de la incubación, centrífuga los gusanos y retire el sobrenadante. Luego agregue 600 microlitros de PBST e incubar las muestras en la oscuridad durante 30 minutos para eliminar el exceso de mancha NR. Después de girar las muestras de nuevo como antes, retire todos menos aproximadamente 50 microlitros de sobrenauta.
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