Mouse anti ki67

Whole brain was fixed in 4 % PFA, embedded in paraffin and serially sectioned at 4 μm in 30 different levels in its horizontal plane, spanning the whole tumor area. Sections were stained with H&E (Sigma-Aldrich, St. Louis MO, USA) and analyzed by a pathologist blinded to experimental groups. Tumor proliferation index was assessed in sections immunostained for Ki67 (mouse anti-ki67, BD, San Jose CA, USA; blocking reagent, M.O.M ImmPRESS kit, Vector Laboratories, Burlingame CA, USA; liquid DAB⁺, Dako North America Inc, Carpinteria CA, USA): 6–9 sections were quantified for each sample, representative of the entire tumor volume. The latter were digitized (Nanozoomer, Hamamatsu, Japan) and analyzed with QuPath 0.2.3 (https://qupath.readthedocs.io/en/latest/), blindly from DGE ²H-MRS results. Thus, the tumor regions on each slide were manually defined with ROIs, followed by semi-automated counting of Ki67^+/- cells to determine the total cell density and the labeling index (% Ki67⁺ cells). Finally, the total cell number for each tumor was estimated based on the total tumor volume (T2-w MRI data), the average cell count per surface area (histologic counting) and assuming a cell radius of 10 µm (as reported in mouse GL261 tumors (Roberts et al., 2020 (link))).

hNPCs were fixed in formaldehyde 3.7% for 15 min at RT and permeabilized in 0.2% Triton X-100 for 15 min Primary antibodies were incubated overnight in 2% BSA at 4 °C, following 40 min of 2% BSA blockage. After washing with PBS, secondary antibodies were incubated for 1 h at RT in the dark. Cells were washed three times with PBS and nuclei were stained with DAPI. Coverslips were mounted on slides using Aqua-Poly Mount (Polysciences) whereas cells on the 384 well plates were covered with glycerol and sealed with AlumaSeal CS (Excel Scientific) for image acquisition in confocal microscopy (Leica) and Operetta (Perkin Elmer), respectively. Primary antibodies used: mouse anti-MAP2 (Sigma-Aldrich), mouse anti-Ki-67 (BD Biosciences), rabbit anti-PAX6 (Santa Cruz Biotechnology), rabbit anti-GFAP (Dako), rabbit anti-γ-H2AX (Cell Signaling Technology), rabbit anti-FOXG1 (Abcam), rabbit anti-DYRK1A (Sigma-Aldrich), rabbit anti-SOX2 (Millipore), mouse anti-β-tubulin III (Millipore), mouse anti-nestin (Millipore), rabbit anti-TBR2 (Millipore). Secondary antibodies used: goat anti-mouse Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific). Data are expressed as relative protein expression in comparison with basal protein expression in control with vehicle (DMSO).

Section immunofluorescence on paraformaldehyde-fixed, paraffin-embedded mouse embryos was performed as described previously [26 ]. Each analysis was performed on at least three independent biological samples. Primary antibodies used were rabbit anti-SOX9 (ref. [3 (link)]) (1:200), goat anti-DDX4 (R&D Systems, RDSAF2030) (1:300), rabbit anti-CYP11A1 (ref. [57 (link)]) (1:300), goat anti-AMH (Santa Cruz, sc6886) (1:300), rabbit anti-FOXL2 (ref. [58 (link)]) (1:300), mouse anti-SYCP3 (Abcam, ab97672) (1:100), rabbit anti-SRY [3 (link)] (1:100), goat anti-GATA4 (Santa Cruz, sc1237) (1:300), mouse anti-Ki67 (BD Transduction Lab, 550609) (1:100) and mouse anti-NEDD4 (BD Transduction Laboratories, 611481) (1:100). All secondary antibodies were purchased from Invitrogen and were used at a dilution of 1:300. These include donkey anti-mouse Alexa 488 (A21202), donkey anti-rabbit Alexa 488 (A21202), donkey anti-goat 546 (A11056) and donkey anti-mouse Alexa 647 (A31571).