Formaldehyde

For a morphological overview of cell nuclei and cell membranes, H&E staining was performed. Subcutaneous tumors and organs (lung, liver, kidney, and spleen) were collected from all animals and fixed in 4% formaldehyde (Carl Roth GmbH, Karlsruhe, Germany) for 24 h and infiltrated in 30% sucrose solution in PBS for 2–3 days. Tissues were snap-frozen in isopentane (Carl Roth) at −60 °C on dry ice. Cryosections with a thickness of 8 µm were obtained using a Leica CM3050S Cryostat (Leica Microsystems, Wetzlar, Germany). The H&E staining was performed for general histology in the automatic staining system (Leica ST5020, Leica Microsystems). The images were acquired using a Keyence microscope (BZ-9000, KEYENCE GmbH, Neu-Isenburg, Germany) at 40-fold magnification, and tumor nucleoli in each section were counted using the cell count function in ImageJ [23 (link)].

At predetermined time points, samples were washed with PBS and fixed in 4% formaldehyde (Carl Roth, Karlsruhe, Germany) for 30 (2D) or 60 min (3D) at room temperature. The samples were then permeabilized with 0.1% (v/v) Triton X-100 (Carl Roth, Karlsruhe, Germany) for 10 (2D) or 30 min (3D), and blocked with 5% goat serum (Sigma-Aldrich, St. Louis, MO, USA) for 30 (2D) or 60 min (3D), respectively. Afterwards, the samples were incubated with primary antibodies (anti-human disialoganglioside GD2, 1:400, BD Pharmingen, Franklin Lakes, NJ, USA; Cleaved Caspase-3 antibody, 1:1000, Cell Signaling, Danvers, MA, USA) at 4 °C overnight, washed three times with PBS, and subsequently incubated with the respective secondary antibodies (goat anti-mouse Alexa Fluor 594, 1:1000, Invitrogen, Carlsbad, CA, USA; goat anti-rabbit Alexa Fluor 488, 1:1000, Invitrogen, Carlsbad, CA, USA) at room temperature for 2 h. Afterwards, samples were again washed with PBS three times prior to nuclear staining with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Sigma–Aldrich, St. Louis, MO, USA) for 60 min. When indicated, F-actin of cells was labeled with phalloidin (Alexa Fluor™ 488 Phalloidin, 1:400, Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature. Stained samples were analyzed by the fluorescence microscopy.

Organotypic cerebellar slice cultures were fixed in 4% formaldehyde (Carl Roth), permeabilized with 1% Triton X‐100 (Thermo Scientific) in PBS for 30‐45 min and blocked with 10% normal goat serum (Sigma‐Aldrich) in 0·2% Triton X‐100 in PBS for 1 h after three washes with PBS. Primary antibodies (anti‐Iba1 Wako, Neuss, Germany; anti‐CD68, Biolegend) were diluted in PBS supplemented with 1% goat serum and 0·2% Triton X‐100, according to the manufacturer's instructions, and were incubated for 1–2 days at 4°C. After three consecutive washes in PBS, OSCs were incubated overnight with secondary antibodies (goat α rabbit/rat‐Cy3, Millipore), diluted in PBS, supplemented with 1% goat serum and 0–2% Triton X‐100 according to the manufacturer's instructions at 4°C in the dark. Images were acquired using a Leica SP8 confocal laser‐scanning microscope (Wetzlar) and analysed with LAS X software (Leica) and ImageJ. Some images were contrast‐enhanced to facilitate visibility in composite figures.