Figure 1.

Ex vivo brain slice spheroid invasion assay workflow. (A) Summary of the ex vivo brain slice invasion assay experimental timeline. (B) Experimental steps required to preform the ex vivo brain slice invasion assay: (i) Whole brains were harvested from 6-week-old male C57BL/6J mice and (ii) the olfactory bulb and cerebellum were removed. (iii) A block of solidified 5% agar was glued to the edge of the vibratome disc directly opposite the disc indentation. (iv) The isolated cerebral cortex was then glued directly to the vibratome disc such that it is leaning against the agar block with the forebrain facing upwards. (v) A Leica vibratome (VT100S) was used to generate brain slices (speed: 0.15 mm/s; frequency: 80 Hz). (vi) The vibratome disc containing the brain was submerged in ice-cold carbogenated slicing solution (95% O₂, 5% CO₂) and (vii) 300 µm brain slices were collected into a beaker containing the cold slicing solution. (viii) Once all brain slices are collected, 2-3 brain slices were transferred onto permeable cell culture inserts submerged in 25% FBS-containing DMEM/F12 medium. (ix) GBM spheroids were generated and (x) implanted onto the brain slices ex vivo. (C) Experimental steps required to generate brain slice z-sections. (i) A brain slice containing GFP-positive GBM spheroids was transferred to a plastic mold and embedded in 4% agar. (ii) Once the agar is solidified, the agar block is removed form the cryomold, the orientation is marked with a sharpie marker, and the agar block is glued to the vibratome disc. (iii) The vibratome was used to generate 200 µm z-sections. Each z-section was collected and mounted onto microscope slices for imaging.