Anti-CD133 Antibody, clone 13A4

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All brains were dissected and post-fixed in 4% PFA overnight at 4 °C, followed by paraffin embedding. Four-micrometer brain tissue serial slices were coronally sectioned by microtome (HM355s, Thermo Scientific, Waltham, MA, USA) and mounted onto glass slides. These sections were used for immunostaining. The slides were deparaffinized and cleared in Clear-Rite™ 3 (6901TS, Thermo Scientific) followed by rehydration in an EtOH gradient. Then the slides were heated to 95 °C in the antigen retrieval buffer (3 g sodium citrate (25114, Sigma-Aldrich), 0.4 g citric acid (251275, Sigma-Aldrich), 1000 mL H2O, pH 6.0) for 30 min, followed by washing with 0.01 M PBS (all washes were performed three times, 5 min each). The slides were permeabilized with 0.3% Triton X-100 (T8787, Sigma-Aldrich, St. Louis, MO, USA) for 20 min, rinsed, and then blocked for 2 h with blocking buffer (5% normal goat serum (G9023, Sigma-Aldrich) and 5% bovine serum albumin (A7096, Sigma-Aldrich)). The samples were incubated with the primary antibodies (Table ​(Table1)1) overnight at 4 °C, washed with 0.01 M PBS, and then incubated with the suitable secondary antibodies. All antibodies are shown in Key resources table. The control samples were incubated in blocking buffer instead without primary antibodies. Nuclei were visualized with mounting medium including DAPI (H-1200, Vector, Burlingame, CA, USA). Images were taken with a Leica SP8 confocal microscope equipped with a × 40 oil immersion lens. The number of immunolabeled cells lining the lateral wall of the lateral ventricle was counted for three sections in each mouse, and at least three animals were used for each experiment.

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