All experimental study protocols were conducted in accordance with institutional, UK home office regulations. MF1 mice were individually heparinized and terminally anesthetized. Once anesthesia was confirmed, surgical procedures for intracardial perfusion were performed for systemic clearance of blood. Thirty milliliters of phosphate buffered saline (PBS) (37°C) were administered at a flow rate of , followed by administration of 40 ml of 4% formaldehyde solution. The brains were extracted and postfixed for 12 h in 4% formaldehyde (4°C), then rinsed in PBS, and sliced to obtain 2 mm sagittal slices. The two central slices close to the midline, one per hemisphere, were used for the light attenuation quantitation study. For each clearing technique, between 6 and 12 mice were evaluated. Tissues were then optically cleared with three procedures. For BABB clearing, samples were dehydrated in methanol (MeOH) for 48 h at room temperature and transferred for clearing in BABB (1:2 mixture of benzyl alcohol and benzyl benzoate) for 48 h. For clearing with pBABB, samples were first treated with a solution known as Dent’s bleach, which comprises a 4:1:1 combination of MeOH, dimethyl sulfoxide (DMSO), and hydrogen peroxide. This solution was applied prior to dehydration with MeOH and clearing with BABB. Dent’s bleach is widely used for whole-mount immunohistochemistry, where hydrogen peroxide is important to block endogenous peroxidase activity and DMSO to enhance tissue penetration,9 but to the authors’ knowledge, it has not previously been used for optical clearing. The samples cleared with CLARITY were prepared according to the passive protocol,7 which aims to remove lipids by passive thermal clearing (37°C) and preserve tissue structure by hydrogel embedding.