Table 1.

Troubleshooting guide for the ex vivo brain slice spheroid invasion assay.

Issue	Possible Causes	Solutions
Large cuts along sides of the brain slices.	Cutting into the side of the brain during removal.	Use curved scissors to cut along the side of the skull pointing outward to avoid the brain.
Brain slices fall apart during transfer steps.	Incorrect placement of the spatula.	Use a spatula with a wide, flat end to scoop up the brain slice. Be sure to place the spatula directly in the center of the slice before lifting.
Fragmented z-sections.	(i) The agar block was glued to the platform at an angle resulting in the blade skipping. (ii) One side of the brain slice is too close to the edge of the agar block. (iii) The z-section is too thin and is falling apart.	(i) Remove the agar block from the platform and re-glue such that the top of the block is perfectly perpendicular to the platform. (ii) Ensure there is sufficient agar on the top and bottom of the brain slice when embedding into agar block. (iii) Increase the thickness of each z-slice.
Spheroid appears too small.	Imaging the edge of the spheroid.	When z-sectioning the brain slice, parts of the same spheroid will appear in multiple z-sections. Thus, if the spheroid appears too small, image the surrounding z-sections to get a more representative sample.
Spheroid is too large and fills the entire z-section of brain slice.	(i) Spheroid too large for the thickness of the brain slice. (ii) Tumour type is highly invasive. (iii) Slice is too thin for the size of the spheroid.	(i) Decrease the initial size of the spheroids. (ii) Decrease the length of the invasion assay. (iii) Increase the slice thickness to 400 µm to create more space.
Spheroid appears out of focus when imaging.	A bubble formed around the spheroid when placing coverslip over z-sections.	Remove the coverslip, blot excess PBS with a Kimwipe™, and replace coverslip to remove bubbles.