Cells were seeded onto 4 well Lab-Tek II chamber slides overnight. Slides were prepared by washing cells twice with PBS (1X) and subsequently fixed with paraformaldehyde (4%) for 10 min. For double-stranded RNA (dsRNA) staining, cells were fixed with methanol (100%) for 5 min at −20°C. Slides fixed with paraformaldehyde (4%) were further incubated with permeabilization buffer (Triton X-100 [0.1%], sodium citrate [0.1%]) for 5 min at room temperature and incubated with blocking buffer (PBS[1X], BSA [0.5%] and goat serum [1%]) for 30 min. Cells were incubated with primary antibody overnight at 4°C, washed three times with PBS (1X) and further incubated with secondary antibody conjugated to Alexa 488, - 594 or −647 (Molecular Probes) for 1 h. Slides were washed three time with PBS(1X) and once with ddH20, and mounted onto glass coverslips using Prolong Gold + DAP1 mounting media (Molecular Probes). Processed slides were imaged using a Zeiss LSM710 confocal microscope and vector profiles analyzed using Zen software (Carl Zeiss).
Prolong gold dap1 mounting media
Manufactured by Thermo Fisher Scientific
Prolong Gold + DAPI mounting media is a ready-to-use solution designed to mount and preserve fluorescently labeled samples for microscopy. It contains an anti-fade reagent and the DNA-binding dye DAPI for nuclear staining.
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2 protocols using prolong gold dap1 mounting media
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Immunofluorescence Staining for dsRNA
2
Immunofluorescence Staining for dsRNA
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Cells were seeded onto 4 well Lab-Tek II chamber slides overnight. Slides were prepared by washing cells twice with PBS (1X) and subsequently fixed with paraformaldehyde (4%) for 10 min. For double-stranded RNA (dsRNA) staining, cells were fixed with methanol (100%) for 5 min at −20°C. Slides fixed with paraformaldehyde (4%) were further incubated with permeabilization buffer (Triton X-100 [0.1%], sodium citrate [0.1%]) for 5 min at room temperature and incubated with blocking buffer (PBS[1X], BSA [0.5%] and goat serum [1%]) for 30 min. Cells were incubated with primary antibody overnight at 4°C, washed three times with PBS (1X) and further incubated with secondary antibody conjugated to Alexa 488, - 594 or −647 (Molecular Probes) for 1 h. Slides were washed three time with PBS(1X) and once with ddH20, and mounted onto glass coverslips using Prolong Gold + DAP1 mounting media (Molecular Probes). Processed slides were imaged using a Zeiss LSM710 confocal microscope and vector profiles analyzed using Zen software (Carl Zeiss).
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