Polyclonal chicken anti gfap

For immunohistochemistry, mice were perfused transcardially with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer after being anesthetized by Zoletil (50 mg/kg) intraperitoneally (Virbac, Carros, France). The whole brain was removed and postfixed in the same fixative for 5–6 h or overnight. The cerebellar vermis was cut into 50-µm sagittal sections. The sections were treated with rabbit monoclonal anti-calbindin D-28 k (1:500, Cloud Clone Corp., China), chicken polyclonal anti-GFAP (1:1000, Abcam, UK), rabbit polyclonal anti-Flag (1:500, Cloud Clone Corp., China) or anti-S100β (1:500, Cloud Clone Corp., China) antibodies and then visualized with Alexa Fluor 594-conjugated donkey anti-mouse t IgG (1:1000, Life Technologies), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:1000, Life Technologies) or Alexa Fluor 647-conjugated goat anti-chicken IgG (1:1000, Life Technologies). The antibodies were dissolved in a PBS solution containing 2% (v/v) normal donkey serum, 0.1% (v/v) Triton X-100, and 0.05% NaN₃. Confocal fluorescence images of the cerebellar slices were obtained from the corresponding region of the cerebellum for comparison. Fluorescent images were obtained using FV10i Confocal Microscope (Olympus, Japan). Images were recorded as Z-stacks using ×10 objective and 1024 × 1024 resolution.