Anti fluorescence quenching sealing agent

The subcellular localization of lncRNA-BIRF was detected by fluorescence in situ hybridization (FISH) [27 (link)]. The lncRNA-BIRF probe and its negative control were synthesized (RIBOBIO, Guangzhou, China), and a fluorescence in situ hybridization kit (RIBOBIO, Guangzhou, China) was used to perform this experiment. Briefly, coverslips containing astrocytes were fixed with paraformaldehyde, and then Triton X-100 was used to permeabilize the cells. Triton X-100 was removed by washing, and the coverslips were sealed with pre-hybridization solution. Subsequently, the coverslips were mixed with probe hybridization solution and incubated overnight at 37 °C. Finally, DAPI staining was performed, and the slices were sealed with anti-fluorescence quenching-sealing agent (Solarbio, Beijing, China) and observed under a laser scanning confocal microscope (OLYMPUS, Japan).

Terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay (KeyGen Biotech, Nanjing, China) was conducted to evaluate apoptosis. Paraffin sections were prepared by conventional methods. After the sections were dewaxed with xylene, hydrated with ethanol gradient, and permeated with proteinase K, the samples were treated with TdT enzyme reaction solution. Finally, streptavidin-fluorescein labeling solution was dropped onto the slide, and the slide was placed into a wet box for incubation at 37 °C for 30 min. We used anti-fluorescence quenching-sealing agent (containing DAPI) to seal the slide (Solarbio, Beijing, China). Finally, we observed and photographed the samples under a microscope (OLYMPUS, DP80, Japan).