Ab66028

Brains were snap frozen in 2-methylbutane cooled in liquid nitrogen and embedded in OCT before cutting. Coronal sections (10 μm) were cut using a freezing sliding microtome (Leica CM1850) and stored at –20°C until use. For S100β and IBA1 immunostaining, slides were washed two times with PBS, blocked and permeabilized with PBS 1% bovine serum albumin, 5% goat serum (Biological Industries, Israel) and 0.05% Triton-X (Sigma-Aldrich) for 1 h and incubated with S100β antibody (1:500, ab66028, Abcam) or IBA1 antibody (1:200, ab178847, Abcam) overnight in 4°C. Slides were washed 3 times with PBS and incubated with secondary goat anti mouse antibody (1/700, Alexa-Flour) for 1 h at room temperature. DNA was stained with DAPI (1:1000, Sigma-Aldrich). For Thioflavin S (ThioS) staining, slides were incubated for 8 min with 0.01% ThioS solution in 50% ethanol. Slices were then briefly incubated twice for 10 s with 80% ethanol, and washed twice with double distilled water (DDW).

The mice, under deep anesthesia, were perfused with 4% paraformaldehyde. Brain sections were cut in the cryostat (14 μm). For immunostaining, the sections were incubated for 1 h in PBS, 0.25% Triton, and 1% BSA. Primary antibodies were incubated overnight at 4°C: rabbit anti-NG2 (1/300, AB5320; Millipore; Saint-Quentin Fallavier, France), mouse anti-GFAP (1/400, MAB360; Millipore), rabbit anti-Olig2 (1/400, AB9610; Millipore), mouse anti-S100β (1/500, AB66028; Abcam, Amsterdam, The Netherlands), mouse anti-adenomatous polyposis coli (APC) (1/600, clone CC1, OP80; Millipore), rabbit anti-Iba1 (1/500, 234003; Synaptic Systems, Uden, The Netherlands), mouse anti-Ki67 (1/200, 550609; BD Pharmigen, Le Pont de Claix, France), and mouse anti-Sox2 (1/100, MAB2018; R&D Systems, Lille, France). The sections were incubated with appropriate secondary antibody (1/200 to 1/400; Millipore, Jackson IR, Montluçon, France) for 2 h at room temperature. Staining was replicated on three or four mice. Images were acquired with a 40X objective (N.A. 0.75) using a fluorescence microscope (Leica DM2000; Leica, Wetzlar, Germany). The images were analyzed and reconstructed with ImageJ 1.39t (Freeware; NIH, New York, NY, United States).

For each animal, six 50 μm coronal brain sections (250 μm between sections) were washed four times, for 10 min in Tris-buffered saline pH 7.4 (TBS) at room temperature, blocked for 1 h in TBS containing 3% horse serum (Millipore; S9135) and 0.3% Triton X-100 (Sigma; 9002-93-1) at room temperature, followed by an overnight staining at 4°C in TBS containing 3% horse serum and 0.3% Triton X-100 with the following primary antibodies: rat anti-BrdU (Abcam, ab6326; 1/200), chicken anti-GFP (Aves, GFP-1020; 1/500), mouse anti-S100β (Abcam, ab66028; 1/100) and rabbit anti-Tbr2 (Abcam, ab23345; 1/300). The next day, brain sections were washed four times for 10 min in TBS at room temperature, blocked for 30 min TBS containing 3% horse serum and 0.3% Triton X-100 at room temperature, and stained for 2 h in TBS containing 3% horse serum and 0.3% Triton X-100 at room temperature with the following secondary antibodies: donkey anti-rat 405 (Abcam, ab175670; 1/400), donkey anti-chicken 488 (Jackson ImmunoResearch, 703-545-155; 1/400), donkey anti-mouse 549 (Jackson ImmunoResearch, 715-507-003; 1/400) and donkey anti-rabbit 647 (Jackson ImmunoResearch, 711-605-152; 1/400). Afterwards, sections were washed four times for 10 min in TBS at room temperature and mounted on glass slides.