Neuronal nuclei (neun)

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).

Enzyme histochemistry was used to observe the glial scar formation (glial fibrillary acidic protein (GFAP) immunohistochemical staining) and motoneurons from lamina IX (Neuronal Nuclei (NeuN) immunohistochemical staining) in the spinal cord tissues. Following three washes in 0.01 mol/L PBS, the slices were incubated with 3% hydrogen peroxide at 37°C for 15 min to block the action of any endogenous peroxidase. Then the sections were incubated with 5% goat serum at 37°C for 30 min. Continually, the tissue sections were incubated overnight at 4°C with GFAP (1:100, rabbit, Bioss) and NeuN (1:100, rabbit, Bioss) primary antibodies respectively. PBS was served as the negative control. Afterwards, sections were separately incubated with PV-9000 reagent 1 (Zhongshan golden bridge) and PV-9000 reagent 2 (Zhongshan golden bridge) for 30 min at 37°C. Subsequently, DAB was incubated as chromogenic agent for 3–7 min in the dark and the sections were counter-stained with hematoxylin. Finally, the positive staining was visualized under an inverted phase contrast microscope imaging system (five sections/animal), the cell area and mean density of GFAP (IOD/area) were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) as above described.