Dylight 488

Briefly, 6-μm-thick sections from the hippoampal DG of the Foxg1 genotype mice (n = 6 in each group) were prepared for immunofluorescence analyses as described previously [12 (link),13 (link)]. To visualize protein expression, sections were first incubated with primary antibodies, including rabbit anti-GFAP (1:200, Wanleibio, cat # WL0836, Shenyang, China), mouse anti-GFAP (1:200, Proteintech, cat# 60190-1-lg, Rosemont, USA), EGFP (1:200, Beyotime, cat # AG281, Shanghai, China), brain lipid binding protein (BLBP) (1:200, Proteintech, cat # 51010-1-AP, Rosemont, USA), Proliferating Cell Nuclear Antigen (PCNA) (1:250, Proteintech, cat # 10205-2-AP, Rosemont, USA), Tbr2 (1:200, Bioss, cat # bs-11331R, Beijing, China), Oligo2 (1:200, Bioss, cat # bs-11194R, Beijing, China), NeuN (1:1200, Bioss, cat # bs-10394R, Beijing, China) and Dcx (1:200, Proteintech, cat # 13925-1-AP, Rosemont, USA). Immunofluorescence secondary antibodies, Dylight 488 (cat # BA1126) and CY3 (cat # BA1032), were obtained from Boster (Wuhan, China). DAPI (KeyGen Biotech, cat # KGA215-50, Shanghai, China) was used to stain the nuclei. After being severally rinsed in PBT, the sections were mounted using antifade mounting medium (Beyotime, Shanghai, China) and fluorescence was visualized using a fluorescence microscope (BX41, Olympus, Tokyo, Japan).

BMSCs were fixed with 4% paraformaldehyde after 7 days of mechanical stretching. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 and then were blocked with 10% goat serum (Solarbio, no. SL038). Primary antibodies for SIRT1 (1:100; Huabio, catalog no. ER130811), LC3 (1:100; ABclonal, catalog no. A17424), and Runt-related transcription factor 2 (Runx2) (1:100; ABclonal, catalog no. A2851) were incubated to detect the relative expression and co-localization of these proteins. Finally, the cells were incubated with secondary antibodies DyLight 488 (1:250; BOSTER, catalog no. BA1045-488) and DyLight 550 (1:250; BOSTER, catalog no. BA1133) and then were photographed using a laser confocal microscope (Zeiss LSM 900). We then used ImageJ (version 1.49) to analyze the fluorescence intensity, which represents the protein expression.