Ab154474

The brain sections on slides were dried at room temperature (RT) for 20 min. The slides were washed twice with PBSTX (1× PBS/0.3% Triton X-100) for 10 min. After incubation with 10 mM sodium citrate for 15 min at 85°C, they were washed with 1 × PBS twice for 5 min and incubated with diluted primary antibody in PBSTX at 4°C overnight. The next day, the slides were washed three times with PBSTX for 10 min at RT, incubated with secondary antibody, and washed three times with 1× PBS for 5 min at RT. Images were obtained using an LSM 880 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany). The primary antibodies are as follows: mouse anti-GFAP (1:500, ab154474, Abcam, Cambridge, United Kingdom), mouse anti-PCNA (1:500, M0879, Dako, Agilent, CA, United States), rabbit anti-S100β (1:500, Z0311, Dako, Agilent), mouse anti-acetylated tubulin (1:250, T6793, Sigma-Aldrich, MO, United States), and rabbit anti-phospho-β-catenin (1:100, D2F1, Cell Signaling Technology, MA, United States). The secondary antibodies are as follows: rhodamine (TRITC) AffiniPure donkey anti-rabbit IgG (1:200, 711-025-152, Jackson Immunoresearch Laboratories, PA, United States) and Cy5 AffiniPure donkey anti-rat IgG (H+L) (1:400, 712-175-150, Jackson Immunoresearch Laboratories). Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; 4083S, Cell Signaling Technology).

For immunofluorescence, the fixed and cryoprotected brains (11 days post-injection survival; n = 8) were serially cryosectioned in the sagittal plane (16 µm section thickness). The serial cryosections were collected as two series (a and b) of alternate sections. Both series were processed for immunofluorescence. Series a was incubated 16 h at 4 °C with a neuronal marker, anti-microtubule associated protein 2 (MAP 2 (2a + 2b); mouse monoclonal antibody clone AP-20, Sigma-Aldrich) diluted 1:200. Series b was incubated 16 h at 4 °C with a radial glia marker, anti-glial fibrillary acidic protein (GFAP; mouse monoclonal antibody [ZRF-1] ab154474, Abcam) diluted 1:1200. After buffer rinses series a was incubated 1 h at room temperature with goat-anti-mouse IgG-Alexa Fluor 488 (Invitrogen A11001) diluted 1:200, and series b with goat-anti-mouse IgG-Alexa Fluor 488 (Invitrogen A11001) diluted 1:200 and the neuronal marker NeuroTrace 640/660 fluorescent Nissl stain diluted 1:100. After buffer rinses the slides were coverslipped with ProLong Antifade Gold with DAPI (Thermo Fischer P36931) as mounting medium.