Paraformaldehyde (pfa)

The LC3-green fluorescent protein (GFP) lentivirus expression vector was produced as previously described (48 (link)). H9C2 cells were infected with LC3-GFP lentivirus in the presence of polybrene (6 µg/ml). Lentivirus-infected cells were selected with blasticidin (5 µg/ml) and maintained for 10 days in medium containing blasticidin. Stably-infected, blasticidin-resistant H9C2 cells were cloned and expanded as previously described (26 (link)). Following different treatments, H9C2 cells were washed three times in PBS and fixed with 4% paraformaldehyde in PBS (Guangzhou Chemical Reagent Factory, Guangzhou, China) for 10 min at room temperature. H9C2 cells were observed at ×650 magnification laser scanning confocal microscopy (IX81 + FV10-MCPSU + IX2-UCB + U-RFL-T; Olympus Corporation, Tokyo, Japan). GFP-LC3 puncta represented the autophagosome. Autophagy was analyzed by quantifying the mean number of GFP-LC3 puncta per cell in all cells in the population by using Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA).

On day 23, mice of each group were intraperitoneally administrated with bromodeoxyuridine (BrdU) (75 mg/kg; Abcam, Cambridge, UK) every 2 hours for 12 hours. Two hours after the last injection of BrdU, the mice were anesthetized with diethyl ether. Blood in the brains was flushed out using normal saline through a heart perfusion, and the brains were fixed using 4% paraformaldehyde (Guangzhou Chemical Reagent Factory, Guangzhou, China), also using heart perfusion. Subsequently, the fixed brains were extracted and continued to fix in a fresh 4% paraformaldehyde for 1 week. The fixed brains were paraffin-embedded and sectioned.
After the denaturation of DNA and antigen retrieval, sections were incubated with a mixture of primary antibody BrdU (1:1400; a marker of newly generated cells) and doublecortin (DCX) (1:400; a marker of immature neurons) (Abcam, Cambridge, UK) at 4°C overnight. The next day, sections were incubated with goat anti-mouse IgG-Alexa 488 (1:600) and goat anti-rabbit IgG-PE (1:400) (Invitrogen, CA, USA) in the dark for 2 hours. DNA in the nucleus was stained by DAPI dye (1:1298) (Boyotime Institute of Biotechnology, Haimen, China) for 5 minutes. Finally, sections were mounted using anti-fade mounting medium, and then stored at 4°C in the dark. A section incubated without primary antibodies was considered as a negative control (Additional Figure 1).

Cytarabine (purity 99.6%, MW 279.68) was purchased from Peking University Pharmaceutical Co., Ltd. (Beijing, China). Egg lecithin (E80) was obtained from Lipoid GmbH (Ludwigshafen, Germany). Sodium Cholesteryl sulfate (SCS, MW 488.7) was obtained from Hubei Saibo Medical Chemistry Goods Limited Company; Octadecylamine (ODA, MW 269.51) was supplied by Sigma International Inc. ε-polylysine (PLL, molecular weight 4000kDa) was purchased from Lanzhou Weiri Bioengineering Limited Company. γ-Polyglutamic acid (PGA, molecular weight 1200kDa) was obtained from Zhejiang Haining City Biotechnology Violet Gold Harbour Limited Company. Sodium sulfite was supplied by Nanjing Longyan Chemical Co., Ltd. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from Beyotime Institute of Biotechnology. Fluorescein isothiocyanate (FITC) was obtained from Sigma International Inc. Paraformaldehyde was purchased from Guangzhou Chemical Reagent Factory. Chromatographic grade methanol was purchased from Concord Technology (Tianjin) Co., Inc. Water was purified by a Barnstead EASYpure^® II RF/UV ultrapure water system (Dubuque, Lowa, USA). All other materials were of analytical grade.