Passive Clarity Brain Tissue Clearing

Mice were perfused with 4% PFA at 45 days after implantation of hG008 cells. For preparation of PASSIVE CLARITY-processed mouse brains, brain tissues were fixed with 4% PFA at 4 °C overnight and then incubated in hydrogel solution (4% PFA, 4% acrylamide, 0.25% VA044 in PBS) at 4 °C for 3 days [24 (link)]. Brain tissues were degassed and polymerized in the same hydrogel solution at 37 °C for 3 h. Four-mm thick coronal sections, except cerebellum and olfactory bulb, were cut. Hydrogel-embedded tissue sections were washed with clearing solution (200 mM sodium borate buffer (pH 8.5) containing 4% SDS) at 37 °C with shaking for 2 h. Sections were then incubated in fresh clearing solution at 48 °C for 5 days. Imaging was performed by multi-photon microscopy (FLUOVIEW FVMPE-RS; Olympus) and 3D images were reconstructed using FV31S-SW software with a maximum intensity projection algorithm (Olympus).