Eg1150h tissue embedder

The formalin fixed tumor samples were processed using the Leica HistoCORE PEARL automated tissue processor, following a 20-hour pre-programmed time-schedule. The tumor samples were wax embedded using the Leica EG1150H tissue embedder. The samples were sectioned into 3 μm sections and were used for hematoxylin and eosin staining (H&E) to assess tissue morphology. Images were acquired on an ImageXpress^® Pico (Molecular Devices) and CellReporterXpress^® software (Molecular devices) and whole-slide acquisition was performed using the 4X objective.

Heads from mice aged 4 weeks were bisected through the midline, the brain removed, and semicircular canal opened, then fixed in 4% paraformaldehyde at RT for 90 minutes. After decalcification in EDTA, the samples were washed for 30 minutes at RT with rotation with the detergent solution (2 mM MgCl₂; 0.02% NP-40; 0.01% sodium deoxycholate; 0.01% sodium deoxycholate in PBS, pH 7.3). X-gal (Promega, cat. no. V394A) was added 1:50 to prewarmed staining solution (5 mM K₃Fe(CN)₆ and 5 mM K₄Fe(CN)₆ in detergent solution), and then the samples were stained at 37°C in the dark overnight. The half-skulls were then washed twice with saline at 4°C in rotation for at least 2 hours and stored at 4°C in 70% ethanol until tissue processing and embedding. The samples were gradually dehydrated in ethanol (Leica TP1020 tissue processor) and embedded in paraffin using xylene as clearing agent (Leica EG1150H tissue embedder). The samples were cut at 8 μm thick, counterstained with Nuclear Fast Red (VWR, cat. no. 342094W), and mounted with Eukitt mounting medium (Sigma-Aldrich). Specimens were imaged using a Zeiss Axioskop microscope connected to an AxioCam camera and interfaced with Axiovision 3.0 software.