Goat anti ki67

The prepared brain slices were washed with 50 mM PBS for 3 times for 10 min each, and then, the slices were incubated in 3% H₂O₂ for 30 min to block endogenous peroxidase activity, followed by immersion in blocking solution for 2h and then incubated overnight with primary antibodies: goat anti-Nestin (1:50, Abcam, UK), goat anti-ki67 (1:100, Abcam UK), goat anti-TrkB (1:100, Abcam UK), mouse anti–NeuN (1:200, Abcam UK), mouse anti–GFAP (1:200, Abcam UK), mouse anti–IBA1 (1:200, Abcam UK). Then, the sections were incubated for 2h at 25°C in the dark with the secondary antibodies: Alexa Fluor 488 goat anti-mouse (1:200, Beyotime, China) for TrkB and ki67; Alexa Fluor 594 IgG goat anti–rabbit (1:200, Beyotime, China) for Nestin, GFAP, IBA1 and NeuN. And then the sections were washed by PBS three times, next wiped the surrounding liquid and re-stained nucleus with DAPI. They were observed by the microscope and analyzed using Image-pro Plus 6.0 software (USA).

Slides were thawed for 10 min at room temperature then rehydrated with 1X PBS. Antigen retrieval was performed by incubating the slides in citrate buffer at 95°C for 15 min. Slides were cooled to room temperature before incubation with 5% NDS in 0.03% Triton X-100 for 1 hour at room temperature and then incubated with goat anti-Ki67 (1:500, Abcam, Cambridge, UK) and mouse anti-doublecortin (Dcx) (1:200, Santa Cruz Biotechnology, Texas, USA) antibodies in 5% NDS in 0.03% Triton X-100 at 4°C overnight. Slides were incubated in secondary antibodies (donkey anti-goat Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 568; 1:400 in PBS, Invitrogen, Oregon, USA) for 1.5 hours at room temperature and then incubated with DAPI (1:10,000, Invitrogen, Oregon, USA) for 10 min. Slides were coverslipped using Vectamount mounting media (Vector, California). Sections were imaged every 200 μm beginning at the crossing of the corpus callosum and ending at the appearance of the anterior commissure. Approximately four to five sections were imaged per brain at a ×20 magnification.