Ab93689

Anesthetized mice were perfused with PBS buffer and then with 4% paraformaldehyde (PFA) in PBS buffer. The brains were then collected in 4% PFA solution, fixed overnight at 4°C followed by the treatment with the sucrose solutions 30% until they sink. The brains were then embedded in optimum cutting temperature (O.C.T.) compound, and sectioned into 10 μm slices on a freezing microtome. Sections were incubated overnight at 4 C with primary antibodies in 3% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20. Primary antibodies used in this study were rabbit anti-Synaptophysin (ab52636, Abcam), rabbit anti-Sox2 (ab93689, Abcam), rabbit anti-Iba1 (17198S, Cell Signaling Technology). Following the overnight primary antibody incubation, sections were washed five times with PBS or TBS buffer solution, incubated with the appropriate secondary antibodies (1:100, Jackson ImmunoResearch Laboratories). For quantification of the expression of Sox2, Sox2-positive cells were counted in the DG of hippocampus. For quantification of the expression of Synaptophysin, the immunofluorescence intensity was quantified using the Image J.

Cell extracts were prepared using RIPA buffer, resolved on NuPAGE 6–12.5% gradient Bis-Tris gels, transferred to nitrocellulose and hybridized using antibodies against p53 (Cell Signaling, 2527S, 1:500), Puma (Abcam, ab9643, 1:500), caspase-3 (Cell Signaling, 9662S, 1:1,000), β-actin (Cell Signaling, 3700S, 1:5,000), H3 (Abcam, ab1791, 1:5,000), H3K9me3 (Abcam, ab8898, 1:1,000), Sox2 (Abcam, ab93689, 1:1,000), H3K4me3 (Abcam, ab8580, 1:1,000), H3K27me3 (Abcam, ab6002, 1:1,000), H3K79me2 (Abcam, ab3594, 1:1,000) and H3K36me3 (Abcam, ab194677, 1:1,000). Uncropped scans of the most important blots are displayed in Supplementary Fig. 10.

At least four sections were analyzed from each tumor. Paraffin-embedded sections were deparaffinized^{51 (link)}, and then incubated with one or more of the following primary antibodies recognizing Sox2 (ab93689), Oct4 (ab18976), Nanog (ab80892), c-Myc (ab125275; Abcam), Ki67 (ab15580) (all from Abcam), cleaved caspase-3 (#9661; Cell Signaling), Bcl-2 (Santa Cruz, sc-65392), or CD133 (MBS462020; MyBioSource) in a solution of PBS with 1% FBS and 0.1% Triton X-100 at 4 °C overnight. Staining was visualized using secondary antibodies tagged with Alexa Fluor 488 (A-21206) or Alexa Fluor 568 (A-11011), both from Thermo Fisher. Nuclei were counterstained using DAPI. Slides were digitally scanned with Panoramic Flash 250 (3DHistech, Budapest, Hungary) using a ×20/0.8 NA objective, and stained cells were counted in five microscopic fields. Images were processed using MetaMorph version 7.8.2 (Molecular Devices).