4 6 diamidino 2 phenylindole dapi

Hippocampi were removed and prepared for dehydration using graded ethanol (70~90%), and immersion fixation in the mixture of dimethylbenzene and wax. Paraffin blocks were then cut into 5 μm sections and all slides were dried in an incubator at 60°C for 2 h, then dewaxed for 30 min and incubated in blocking buffer for 30 min at room temperature (20~24°C). Next primary antibodies against glial fibrillary acidic protein (GFAP, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and BCL2L1, MMP-9 and CC3 (1:100; Bioss Technology, Beijing, China) were added and sections were incubated at 4°C overnight. After multiple rinses in phosphate-buffered saline, the sections were blocked with goat anti-mouse IgG and goat anti-rabbit IgG (1:200; Beyotime Institute of Biotechnology, Shanghai, China) and all the sections were stained with 4,6-diamidino-2-phenylindole (DAPI, Biosharp, Carlsbad, CA, USA) at room temperature (20~24°C). For double immunofluorescent analysis, the sections were imaged and photographed using a laser scanning confocal microscopy (Olympus, Tokyo, Japan).

Spinal cord sections (5-μm thick) from each specimen were deparaffinized with xylene (2 ​× ​20 ​min), incubated in graded concentrations of ethanol (2 ​× ​anhydrous ethanol, 95%, 90%, 80%, and 70%), and washed thrice in phosphate-buffered saline (PBS). The sections were incubated in H₂O₂ for 15 ​min to block endogenous peroxidase, with a blocking solution (0.1% Triton X-100 in PBS and 10% goat serum) at room temperature for 1 ​h and finally with the primary antibodies overnight at 4 ​°C. The primary antibodies used were as follows: mouse anti-NeuN antibody (1:200, ab104225; Abcam, MA, USA) and goat anti–rat glial fibrillary acidic protein (GFAP) antibody (1:400, ab53554; Abcam, MA, USA). The sections were washed thrice with PBS and then incubated with secondary antibodies for 1 ​h at room temperature. After 3 rinses in PBS, the nuclei were counterstained with 1 ​g/ml 4′,6-diamidino-2-phenylindole (DAPI) (Biosharp, Hefei, China) for 5 ​min. The anti-fade mounting medium (Solarbio, Beijing, China) was placed on each slide. Immunofluorescence imaging was visualized using an Olympus fluorescence microscope (TH4-200; Olympus, Tokyo, Japan), and quantification was performed using ImageJ software (National Institutes of Health, US).

All standard synthesis reagents were purchased from commercial suppliers and used without any further purification. ZrOCl₂·8H₂O, 2,5-dibromobenzenamine, 4-(methoxycarbonyl) phenylboronic acid, CsF, Pd(PPh₃)₄, RITC, and FITC were obtained from Energy Chemical. NaOH and CH₃COOH were purchased from Sinopharm Chemical Reagent. INS, Tf, 4′,6-diamidino-2-phenylindole (DAPI), lysosome tracker, and the BCA kit were obtained from Bio-sharp (Hefei, China). MTT and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Wuhan Kerui Biotechnology Co. Ltd. The bovine INS ELISA kit was obtained from Jiangsu Meimian Industrial Co. Ltd.