Ribotm fluorescent in situ hybridization kit

GC cells were fixed with 4% formaldehyde for 15 minutes, washed with PBS, treated with pepsin, and dehydrated with ethanol. According to the manufacturer's instructions, RNA fluorescence in situ hybridization (FISH) was performed using a RiboTM Fluorescent In situ Hybridization Kit (RiboBio, China). The 4',6-diamino-2-phenylindole (DAPI) was performed to stain DNA. The GFP-labeled TP73-AS1 probe was detected and observed by Leica-SP8 confocal microscope (Leica, Germany).

Fluorescence in situ hybridization was performed as described previously (Guo et al., 2018 (link)). CFs were rinsed in 1 x PBS and then fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were rinsed in 1 x PBS at 4°C. 200 ml of Pre-hybridization Buffer was added at 37°C for 30 min. Hybridization was carried out with a FISH probe (Ribo^TM lncRNA 554 FISH Probe Mix (Red)) at 37°C in the dark overnight using Ribo^TM Fluorescent In Situ Hybridization Kit (C10910, RiboBio). The cells were washed with Wash Buffer I, II, and III at 42°C in the dark. The cells were stained with DAPI in dark and then washed with 1 x PBS three times. All images were obtained with a fluorescence or confocal microscope (LSM880; Zeiss, Jena, Germany).

Fluorescence in situ hybridization assay was used to ascertain the cellular localization of miR-199a-5p and LINC00319. An RNA FISH probe mix for LINC00319 with Cy3-labeling and a FISH probe mix for miR-199a-5p with FAM-labeled were each designed (GenePharma, China), and applied to HSC6 cells using a RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) according to kit instructions. HSC6 cells were plated in 24-well culture plates at a concentration of 6 × 10⁴ per well. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with a pre-hybridization buffer. In all, 4 μmol/L FISH probes for miR-199a-5p and LINC00319 were added on HSC6 cells with the hybridization buffer and incubated at 37 °C overnight. Thereafter, cell nuclei were cunterstained with 4,6-diamidino-2-phenylindole (DAPI) and the images were obtained with laser scanning confocal microscopy (Carl Zeiss AG, Germany).

FISH assays were performed to detect RNA levels using Ribo^TM Fluorescent in Situ Hybridization Kit (RiboBio) according to the manufacturer’s protocol. The Cy3-labeled probes complementary to U6, 18 S rRNA, or the back-splice site of circITGB6 (Supplementary Table 5) were used for hybridization, and the signal was detected with a confocal laser scanning microscope (Zeiss LSM510).

The expression of miR-106b-5p in 5 human malignant melanoma samples and adjacent normal tissues was analysed by FISH. FISH was carried out by using a RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China) as previously described [23 (link)]. Nuclear signals and all signals in the positive sites of the miR-106b-5p probes were counted in different fields of view of the tissue, and 3 fields of view were selected for each group. The FISH results were calculated as follows: FISH result = probe positive signal / nuclear signal. ImageJ software was used to collect the signals.

To detect the subcellular location of LAMP5-AS1 RNA on LAMP5 locus, we carried out FISH in THP1 cells using the RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China). Cells were washed briefly with PBS and then fixed in 4% formaldehyde for 15 min at room temperature. Cells were permeabilized in PBS containing 0.5% Triton X-100 on ice for 5 min and then blocked in the preliminary hybridization solution for 30 min at room temperature after three washes with PBS for 10 min each. Hybridization was carried out using the Cy3-labeling LAMP5-AS1 FISH Probe Mix (RiboBio, China) and Digoxigenin (DIG) -labeling LAMP5 DNA primers (the DNA primers were labeled DIG using the DIG High Prime DNA Labeling and Detection Starter Kit II, Roche, Switzerland) in a humidified chamber at 37 °C for 12–16 h. Cells were rinsed with SSC buffer in accordance with the order 4×, 2×, and 1×. For co-localization studies, after RNA and DNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with DOT1L primary antibody (CST, 90,878 S) and followed by DIG-FITC (Abcam, ab119349) and Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115). Finally, cells were observed on a Zeiss7 DUO NLO confocal laser microscope (Carl Zeiss, Germany).