4 6 diamidino 2 phenylindole dapi staining

The apoptosis of cortical neurons was determined using an in situ terminal deoxynucleotidyl-transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling assay (TUNEL, Roche Diagnostics, Germany). Briefly, rat brain tissues in cortex region were fixed in 4% paraformaldehyde, cut into 14 μm coronal sections and stained as the manufacture’s instruction. Then, the sections were immerged into 4′, 6-diamidino-2-phenylindole (DAPI) staining (Beyotime Biotechnology, China). TUNEL-positive (green) and DAPI-positive (blue) staining patterns were acquired under a fluorescence microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan).

Briefly, the fixed cells were permeabilized and incubated with anti-PTBP2 antibody, anti-human STAT1 antibody (#10144-2-AP, Proteintech, Chicago, IL, USA), and anti-IRF9 antibody (#14167-1-AP, Proteintech, Chicago, IL, USA) separately. They were further incubated with anti-mouse or rabbit Alexa Fluor secondary antibodies (#4414 or #4409, Cell Signaling Technology, Beverly, USA). The nuclei were subsequently visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining (#P0131, Beyotime, Nanjing, China). Images of the cells were captured by a confocal microscope (Leica SP8, Germany) equipped with a 20× objective and then analyzed using Image-Pro Plus 6.0 software.

NF-κB was detected by using the NF-κB Activation Nuclear Translocation Assay Kit (Beyotime, Jiangsu, China), and the nucleus was identified by 4′,6-diamidino-2-phenylindole (DAPI) staining (Beyotime).