2 methylbutan

Fresh whole brains of single/double knockouts and respective controls were removed from the skull as described above. To obtain sagittal sections, hemispheres were separated with a cut along the midline and placed medial side down on a flat piece of thin acryl glass (Geisler et al., 2019 (link)). Subsequently, mounted hemispheres were submerged for 1 min in 2-methylbutan (Carl Roth, Karlsruhe, Germany) cooled to −50°C. Frozen samples were stored in sealed vials at −80°C until further processing and transferred to −20°C 1 day before sectioning. Brain samples were mounted on a tissue holder using Tissue-Tek^® O.C.T.^TM Compound (A. Hartenstein, Würzburg, Germany). Consecutive sections (20 μm) of one hemisphere were obtained with a cryotome (NX50: Histocom, Vienna, Austria), collected on polysine coated glass slides (Lactan, Graz, Austria), and stored at −20°C until further use.

Spheroids were fixed with 4% PFA for 15 min, embedded in Tissue-Tek O.C.T. (Sakura Finetek, Germany), and snap-frozen in cold 2-methylbutan (Carl Roth, Germany). Cryosections (10 µm) were obtained using a Leica JUNG CM3000 cryostate (Leica Microsystems, Germany) and transferred on SuperFrost slides (Thermo Fisher Scientific, USA/MA). Sections were immersed in PBS, permeabilized with 0.1% triton/PBS, and blocked with 5% goat serum. Samples were stained overnight at 4 °C with primary rabbit α-CD31 (Invitrogen, USA/MA, 1:100) and mouse α-PSMA (Abcam, UK, 1:250) antibodies. After washing, sections were incubated with α-mouse Alexa Fluor 488 (Thermo Fisher Scientific, USA/MA, 1:1000) and goat α-rabbit Alexa Fluor 555 (Cell Signaling, USA/MA, 1:1000) antibodies for 1 h at room temperature. DAPI staining was applied for 3 min and the cells were mounted with Mowiol. Images were acquired with a Zeiss Imager Z.1 microscope (Zeiss, Germany).