Lncrna fish probe mix

Fluorescence in situ hybridization (FISH) was used to detect the localization of LINC00491 in HUH-7 cells. The lncRNA FISH Probe Mix (RIBOBIO) was used. The HUH-7 cells were trypsinized and re-suspended in fresh media to a final concentration of 5 × 10⁴ cells/ml. Then 400 μl/well of the cell suspension was added to a 24-well plate and incubated for 24 h. Then the cells were washed with PBS and fixed with 4% paraformaldehyde. Subsequently, the cells were blocked with 0.5% Triton X-100, incubated with prehybridization solution, and hybridized with LINC00491 probe overnight at 37 °C. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) and the cells were observed under a confocal laser microscope (Leica, Mannheim, Germany).

For FISH assay, LINC00460 fluorescent probe was accessed from Ribo Co., Ltd. (China). According to instructions, Ribo Fluorescent in Situ Hybridization Kit and Ribo lncRNA FISH Probe Mix were employed for FISH assay (Ribo, China). Fluorescence microscope was utilized for observation and photographs. Nuclear-cytoplasm separation in HGC27 or SNU-1 cells was completed with PARIS Kit (Life Technologies, USA), and then qRT-PCR assayed levels of LINC00460 and reference genes (U6 and GAPDH) in nucleus and cytoplasm.

The FISH Kit and lncRNA FISH Probe Mix (Ribo, Guangzhou, China) were used for FISH assays following the manufacturer's instructions (Guangzhou RiboBio Co., Ltd., Guangzhou, China). Briefly, cells seeded onto the slides in 24-well culture plates were fixed in 4% paraformaldehyde at room temperature for 30 mins. Cells were then cultured with a lncRNA probe and then counterstained with DAPI. Finally, cells were observed and images were captured on the laser scanning confocal microscope (Olympus, Tokyo, Japan). In this experiment, the researchers used U6 and 18S RNA as the controls for the nucleus and cytoplasmic subcellular localization, respectively.

LncRNA FISH Probe Mix (RiboBio, Guangzhou, China) was used to perform the FISH assay. Briefly, fibroblasts were fixed with 4% paraformaldehyde (Solarbio) and treated with protease K, glycine, and acetylation reagents. After prehybridization at 37°C for 30 min, fibroblasts were hybridized with Cy3-labelled HSFAS probes at 37°C overnight. Then, the fibroblasts were incubated with anti-ADAMTS8 antibody (Santa Cruz, Santa Cruz, USA) for 2 h at 37°C, and then FITC-conjugated secondary antibodies (Absin, Guangzhou, China) were used for signal visualization according to the standard protocols of immunofluorescence staining
[19] (link). The nuclei were stained with DAPI. A confocal laser-scanning microscope (Zeiss, Oberkochen, Germany) was used to scan the images.

LncRNA FISH Probe Mix (RiboBio, Guangdong, China) was used to perform the FISH assay. Briefly, chondrocytes were fixed with 4% paraformaldehyde and treated with protease K, glycine, and acetylation reagents. After prehybridization at 42°C for 1 hour, chondrocytes were hybridized with Cy3‐labeled H19 probes and 488‐labeled miR‐29b‐3p at 42°C for 12 hours. The nucleus was stained with DAPI. 18S and U6 were internal controls in which 18S was distributed in the cytoplasm and U6 in the nucleus.

The cells on the cover clips were fixed with 4 % cold paraformaldehyde. After two washes in PBS, the cell membranes were permeabilized with 0.1 % TritonX-100. For IF, the cells were washed with PBS and blocked with 5 % BSA for 1 h followed by incubation with anti-VEGFA (1:250, Abcam, USA) at 4 °C overnight and then incubation with secondary antibodies conjugated to fluorescein isothiocyanate (1:200, EarthOx, USA) at room temperature for 40 min. The nuclei were dyed with 40,6-diamidino-2-phenylindole (DAPI). For FISH, lncRNA FISH Probe Mix and Fluorescence In Situ Hybridization Kit (RIBO Bio, China) were used for detecting TPT1-AS1 expression in CRC cells following the manufacture's protocol. Photos were obtained through laser scanning microscopy (FV1000, Olympus, Japan).