Methyl β cyclodextrin

The schedule for tissue clearing with ScaleSF is described in Figure 2A. Brain slices were permeabilized with ScaleS0 solution for 2 hr at 37°C, washed twice with PBS(–) (27575-31, Nacalai Tesque) for 15 min at 20–25°C, and cleared with ScaleS4 solution for 8–12 hr at 37°C. We treated brain slices with ScaleS4 solution for 12 hr in the data shown in this paper. The formula for ScaleS0 solution was 20% (w/v) sorbitol (06286-55, Nacalai Tesque), 5% (w/v) glycerol (G9012, Sigma-Aldrich), 1 mM methyl-β-cyclodextrin (M1356, Tokyo Chemical Industry), 1 mM γ-cyclodextrin (037-10643, Wako Pure Chemical Industries), and 3% (v/v) dimethyl sulfoxide (DMSO) (13407-45, Nacalai Tesque) in PBS(–), and that for ScaleS4 solution was 40% (w/v) sorbitol, 10% (w/v) glycerol, 4 M urea (35940-65, Nacalai Tesque), 0.2% (w/v) Triton X-100 (35501-15, Nacalai Tesque), and 25% (v/v) DMSO in distilled deionized water (DDW) (Miyawaki et al., 2016 (link)).
The optical clearing methods of brain slices with CUBIC, PACT, ScaleSQ(0) and SeeDB followed the protocol as below. Considering 1-mm-thick slices clearing, incubation time of each method was adjusted accordingly.

Transparent mouse brains were generated by the ScaleS method as previously described23 (link). In brief, ScaleS solutions were made using urea crystals (Wako Pure Chemical Industries, 217–00615), D(−)-sorbitol (Wako Pure Chemical Industries, 199–14731), methyl-β-cyclodextrin (Tokyo Chemical Industry, M1356), γ-cyclodextrin (Wako Pure Chemical Industries, 037–10643), N-acetyl-L-hydroxyproline (Skin Essential Actives, Taiwan), dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, 043-07216), glycerol (Sigma, G9012), and Triton X-100 (Nacalai Tesque, 35501-15). A mouse brain of a human mutant APP knock-in mouse22 (link) was fixed at indicated ages and cleared with ScaleS. For immunohistochemistry, the following fluorophore-conjugated antibodies (Abs) were used: mouse monoclonal antibody to amyloid-β conjugated to Alexa Fluor-488 (Covance; SIG-39347, 1:200), rat Ab to pSer46-MARCKS conjugated to CF633 (Biotium) and goat synapsin-1 antibody conjugated to Oyster 550 (Luminartis). The images were acquired with a TPEFM system (Olympus FVMPE-RS) with 920-nm excitation and an objective lens (XLSLPLN25XGMP) with a numerical aperture (NA) = 1.0, working distance (WD) = 8 mm, refractive index (RI) = 1.41–1.52, a collection collar, and a z-drive. The z step was 2.0 μm.