Ribo lncrna fish probe mix red kit

Based on the protocols in the RiboTM lncRNA FISH Probe Mix (Red) Kit (Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China), fluorescence in situ hybridization (FISH) was operated to identify the subcellular location of PGM5‐AS1 expression in osteosarcoma cells. The osteosarcoma cells were positioned onto the slide in a 6‐well plate, followed by 1‐h culture till the cell confluence was about 80%. After fixation with 1 mL of 4% paraformaldehyde, the cells were manipulated with protease K (2 μg·mL⁻¹), glycine, and acetylation reagent, and then incubated with 250 μL prehybridization. After the removal of the prehybridization, the cells were hybridized overnight with 250 μL hybridization solution supplemented with 300 ng·mL⁻¹ probe. The fluorescence microscope (Olympus Optical, Tokyo, Japan) was applied for the observation of cells.

Bioinformatics tools⁴ (last accessed date: November 13, 2018) were used to obtain information about the subcellular localization of LINC01315. Then, as described previously (21 (link)), we used the fluorescent in situ hybridization (FISH) for verification, following the instructions provided in the Ribo^TM lncRNA FISH Probe Mix (Red) kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China). SAS cells were seeded in a six-well plate for 1 day to attain cell confluence of about 80%. On the next day, the cells were fixed with 4% paraformaldehyde (1 mL) at room temperature, followed by treatment with proteinase K (2 μg/mL), glycine, and acetylation. The cells were then incubated with 250 μL of pre-hybridization solution at 42°C for 1 h and subsequently incubated with 250 μL of 300 ng/mL hybridization solution containing the probes overnight at 42°C. On the next day, the cells were stained for 5 min in a 24-well plate with Hoechst solution diluted in phosphate buffered saline tween-20 at the ratio of 1:800. Before observation, cells were mounted in anti-fluorescence quencher. Five different randomly selected fields were observed and photographed under the fluorescence microscope (Olympus, Tokyo, Japan).

The subcellular localization of LINC00261 was determined by FISH analysis as explained previously [56 (link)], following the instruction of Ribo^TM lncRNA FISH Probe Mix (Red) Kit (C10920, RiboBio, Shenzhen, China). Briefly, the cell slide was seeded in a 24-well plate at a density of 6 × 10⁴ cells/well until the cell confluence reached 60–70%. Subsequently, cells were fixed by 1 mL 4% paraformaldehyde at room temperature for 10 min and permeabilized with 1 mL precooled phosphate buffer saline (PBS) containing 0.5% Triton X-100 at 4 °C for 5 min. After PBS washing, cells in each well were sealed with 200 µL prehybridization solution at 37 °C for 30 min, and then hybridized with nucleotide probe against LINC00261 (GeneCreate, Wuhan, China) at 37 °C overnight without light. Cells were rinsed in Lotion I (4 × SSC, 0.1% Tween-20), Lotion II (2 × SCC), Lotion III (1 × SCC), respectively, and then washed with 1 × PBS three times (5 min for each). Finally, cells were stained in 4′,6-diamidino-2-phenylindole solution (1: 800) for 10 min, washed, and sealed with nail enamel. Five visual fields selected at random, cells were observed and imagined under a fluorescence microscope (Olympus, Japan).