Bm0055

Brain tissues were cut into frozen sections. The frozen sections were air-dried at room temperature, washed with PBS, permeabilized with 0.4% Triton X-100, and submitted to antigen retrieval as for immunohistochemistry. After washing with PBS, sections were blocked in normal goat serum for 1 h. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: anti-ALK5 (1:50, SAB4502958, Sigma-Aldrich, USA), anti-neuron-specific beta-III tubulin (1:50, MAB1195, R&D, USA), anti-GFAP (1:100, BM0055, Boster, China), anti-nestin (1:100, ab11306, Abcam, USA), and anti-DCX (1:50, sc-271390, Santa Cruz Biotechnology, USA) antibodies. The next day, the sections were washed with PBS and incubated with a mixture of goat anti-rabbit IgG-CFL 488 (1:100, sc-362262, Santa Cruz Biotechnology, USA) and goat anti-mouse IgG-CFL 555 (1:200, sc-362267, Santa Cruz Biotechnology, USA) at 37 °C for 1 h in the dark. Then, the sections were counterstained with DAPI for nuclei. Images were captured by a confocal laser scanning microscope (A1 + R, Nikon, Tokyo, Japan).

Brain tissues were fixed, dehydrated, and then cut into coronal sections at a thickness of 10 μm. Sections were permeabilized with 0.3% Triton X-100. Then, brain slices were blocked with 5% donkey or goat serum for 1 h and incubated overnight at 4°C with primary antibodies: ABIN1 (bs-9568R, Bioss, China, 1 : 50), NeuN (MAB377, Millipore, Germany, 1 : 200), A20 (3A11G6, Proteintech, USA, 1 : 100), Iba-1 (NB100-1028, Novus, USA, 1 : 50), and GFAP (BM0055, Boster, China, 1 : 100). Then, the following fluorescently labeled secondary antibodies were incubated with the sections at 37°C for 1 h in the dark: CoraLite594-goat anti-rabbit (SA00013-4, Proteintech, 1 : 200), CoraLite488-goat anti-mouse (SA00013-1, Proteintech, 1 : 200), CoraLite594-donkey anti-rabbit (SA00013-8, Proteintech, 1 : 200), FITC-donkey anti-goat (SA00003-3, Proteintech, 1 : 200), and Cell nuclei were stained with DAPI. Brain slices were observed under a laser confocal microscope (LSM-800, Carl Zeiss Micro-Imaging Co., Germany).