Radius image acquisition software

LV wholemount and coronal IHC preparations were visualized using a Zeiss LSM880 confocal microscope. For coronal sections, the entirety of the coronal SVZ was imaged dorsal to ventral on sections containing tumor or injection site. Images were taken using 10X, 20X, 40X, or 63X objectives. SEM images were obtained on a Hitachi S4800 microscope. TEM images were obtained with a FEI Tecnai Spirit G2 biotwin microscope with a Xarosa (20 Megapixel resolution) digital camera using Radius image acquisition software (EMSIS GmbH, Münster, Germany). ZEN Blue (Zeiss), ImageJ (NIH), and Vision4D (arivis) were used for image processing and quantification.

Samples were post-fixed with a solution of 1% osmium tetroxide and 7% glucose in 0.1 M PB for 30 min at room temperature, washed with deionized water and partially dehydrated in increasing concentrations of ethanol. Then, sections were contrasted with 2% uranyl acetate in 70% ethanol for 2 h 30 min at 4°C. Subsequently, dehydration with 70%, 96%, 100% ethanol, and propylene oxide was carried out. Sections were embedded in Durcupan epoxy resin (Sigma-Aldrich), where they remained overnight at room temperature in gentle shaking. The next day, samples were transferred to flat molds with fresh resin followed by 72 h at 70ºC to resin polymerization. Once the resin had polymerized, the region of interest (zone with BCAS1⁺ cells) in each section was selected and semithin sections (1.5 μm) were obtained using a UC7 ultramicrotome (Leica). After staining the sections with 1% toluidine blue solution, ultrathin sections (70–80 nm) were obtained. Finally, images were taken at 80 kV on a FEI Tecnai G² Spirit transmission electron microscope (FEI Company, Hillsboro, OR) equipped with a Xarosa (20 Megapixel resolution) digital camera using Radius image acquisition software (EMSIS GmbH, Münster, Germany).