Diamidino 2 phenylindole dapi

Animals were anesthetized and transcardially perfused with 0.01M PBS and 4% paraformaldehyde (PFA) seven days after HI. The brains were immediately removed and post-fixed in 4% PFA. After dehydration with a sucrose gradient, 20 serial coronal sections were cut across the middle hemisphere. Sections were then washed thrice with 0.01M PBS, blocked with 5% bovine serum albumin (BSA), and used for active neuronal nuclei (NeuN) and microtubule association protein-2 (MAP2) staining. Eventually, the sections were incubated overnight at 4 °C with anti-NeuN (1:300 dilution, Abcam, ab177487) and anti-MAP2 (1:200 dilution, Proteintech,17490-1-AP) primary antibody. After three washes in 0.01M PBS, the sections were incubated with Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:2000 dilution, Boster Biological Technology, BA1032) for one hour at room temperature. After three washes in 0.01M PBS, they were finally covered with diamidino-2-phenylindole (DAPI, 1:1000, Beyotime, C1002) for five minutes. For each staining, five non-overlapping digital microscopic images of the hippocampal areas were captured randomly using a fluorescence microscope (IX71, OLYMPUS, Japan), and the number of positive cells in the hippocampal CA1 area was distinguished via Image-Pro Plus 6.0.

Cultured hADSCs were fixed with 4% paraformaldehyde for 15 min at RT, permeabilized with 0.1% Triton X-100 for 5 min at RT, washed three times with PBS, and blocked with 5% bovine serum albumin (BSA) in TBST for 30 min. hADSCs were incubated with rabbit polyclonal antibody against β-catenin (1:200) overnight at 4°C and then washed 3 times with PBS. hADSCs were incubated with FITC-conjugated goat anti-mouse IgG (H+L) (1:200, Beyotime Biotechnology, China) for 2 h at RT and washed 3 times with PBS, and the nuclei were stained with diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology, China). The intensity of immunofluorescence was analyzed by Fiji ImageJ software.

IF staining was performed using treated SH-SY5Y cells on poly-lysine‐coated glass coverslips. Cells were washed twice with pre-cooled PBS and then fixed with 4 % paraformaldehyde for 30 min. Afterwards, cells were permeabilized with 0.2 % Triton X-100 in PBS at room temperature for 5 min, and washed twice with PBS. Non-specific binding sites were blocked by 0.5 % bovine serum albumin (BSA) for 30 min. After three further washes with PBS, cells were incubated overnight at 4 °C with anti-VP1 (For CV-A16, 1:1000 dilution; Millipore, USA) or anti-VP1 (For CV-A10, 1:1000 dilution; GeneTex, China) and anti-Caspase1 (1:100 dilution; Affinity, UAS), followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG and Alexa Fluor 594-conjugated donkey anti-rabbit IgG secondary antibodies (1:300 dilution; CST, USA), for 2 h at room temperature. After three final washes, the nuclei were counterstained with diamidino-2-phenylindole (DAPI; 1:1000 dilution; Beyotime, China) for 5 min, and then washed three more times with PBS. Finally, the cells were analyzed using a confocal laser scanning microscope (Leica, Germany).