Fluorescence in situ hybridization (FISH) was performed with a FISH kit (Ribobio Co.) according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4×, 2×, and 1× SSC, the cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Finally, the samples were observed using a BX60 fluorescence microscope (Olympus, Tokyo, Japan).
IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3- or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit or goat anti-mouse IgG (Sangon, Shanghai, China) was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope (Olympus).
IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3- or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit or goat anti-mouse IgG (Sangon, Shanghai, China) was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope (Olympus).