Sample preparation
All animal experimental procedures were conducted in accordance with KBRI IACUC guidelines. Adult Thy1 YFP mice (20 weeks old) were anaesthetized with Avertin (250 mg/kg, Sigma) and perfused transcardially with 30 mL PBS, followed by 20 mL fresh 4% PFA (10 mL/min). Brains were extracted and incubated in 4% PFA for 24 hours at 4 C. Brains were then washed and stored in PBS for 24 hours at 4 C. Hydrogel infusion and polymerization A hydrogel solution was prepared using the X CLARITY ™ Hydrogel Solution Kit (Logos Biosystems, C1310X). Brains were incubated in hydrogel solution (5 mL/brain) for 24 hours at 4 C. Brains in solution were placed in the X CLARITY™ Polymerization System (Logos Biosystems, C20001) for 2 hours at 37 C and 90 kPa to initiate polymerization. Electrophoretic Tissue Clearing The hydrogel embedded brains were rinsed with Electrophoretic Tissue Clearing Solution (Logos Biosystems, C13001) and then placed in the ETC Chamber of the X CLARITY ™ Tissue Clearing System (Logos Biosystems, C10001). Electrophoretic Tissue Clearing Solution was circulated through the chamber and 0.7 A was applied across the brains for 6 12 hours at 35 C. After clearing, brains were washed with PBS overnight at room temperature to remove residual SDS.

Technical Tips:
• Post fixation is an important step in the X CLARITY ™ protocol. A whole mouse brain should be post fixed for at least 24 hours. Excessive fixation can lead to increased clearing time.
• Post fixed tissues should be washed for at least 24 hours as residual PFA can react with acrylamide during hydrogel polymerization and lead to increased clearing time.