Tissue Clearing and Staining

Slightly modified CUBIC and PACT tissue clearing protocols^43,47 were used as follows for brain tissue clearing, with the exception of the Sorafinib treated brains. Following 24 hours fixation, brains were rinsed twice with 1 × PBS, sliced into 2 mm sagittal sections, and incubated in a hydrogel formulation (2 or 4% acrylamide in 1x PBS with 0.25% photoinitiator VA044 [Wako Chemicals]) at 4 °C gently rocking for three days. Prior to hydrogel polymerization, samples were degassed using a desiccation chamber, alternating between three 10-minute cycles of vacuum and nitrogen gas. Samples were polymerized by incubation at 37 °C for 3–4 hours. Polymerized hydrogel was decanted and samples were rinsed twice with 1 × PBS. Lipid removal from samples was then performed using either 8% SDS in 1 × PBS. Once samples became optically transparent after approximately 4–7 days, samples were washed for 24 hours in 1 × PBS before whole tissue staining (molecular phenotyping). For molecular phenotyping, all samples were first incubated in a 1:50 primary antibody dilution (anti-Ms mAb GFAP (GA5), Cell Signaling Technology Technologies^®, Cat. No. 3670s; anti-Rb mAb Cytokeratin 8 (EP1928Y), Abcam^®, Cat. No. ab53280) in 0.1% Triton X-100 for 3 to 7 days with gentle rotating. Following primary antibody incubation, samples were washed for 24 hours in 0.1% Triton X-100 with minimum three rounds of buffer changes. All samples were then incubated in a 1:50 secondary antibody (goat anti-Rb or goat anti-Ms Alexa Fluor^® 488, 594, or 647) dilution in 0.1% Triton X-100 for 3 to 7 days with gentle rotating. After secondary antibody incubation, EdU was detected per Click-iT EdU Alexa Fluor^® 488 or 594 imaging kit (Life Technologies™, Cat. No. 10639) protocol with a 3–4 hour detection incubation period. Some samples were incubated in DAPI (0.025 mg/mL) for 12 hours with gentle rotating. Samples were washed one last time in 0.1% Triton X-100 for 24 hours with multiple buffer changes and then made optically transparent by incubation in CUBIC 2 reagent prepared as described previously⁴³ for at least 2 hours prior to imaging. The brains of Sorafinib treated mice were cleared using ScaleAB methodology as previously described⁴⁴.