Supplementary Figure 1: Experimental protocols for ScaleA2, ScaleS, AbScale, ChemScale, and ScaleSQ

Tissue clearing protocols that use ScaleA2, ScaleS, and ScaleSQ solutions, and AbScale and ChemScale clearing/labeling protocols with relevant theoretical background.

In each protocol, a brain sample is incubated in a large volume (> 25 ml/g tissue) of the solution.

(a) ScaleA2 protocol. See the original paper (Ref. 2) for details.

(b) ScaleS protocol. The formulas of ScaleS solutions are shown in Supplementary Table 1. This procedure assumes that an adult mouse cerebral hemisphere is processed as a sample to be cleared. When mouse brain slices are used, the incubation time for each step can be substantially shortened.

First, permeability of a sample is enhanced by incubation in ScaleS0 solution containing 20% sorbitol, 5% glycerol, 1 mM methyl-β-cyclodextrin, 1 mM γ-cyclodextrin, 1% N-acetyl-L-hydroxyproline, and 3% DMSO for 12 hrs. Methyl-β-cyclodextrin and γ-cyclodextrin extract cholesterol from biological membranes, whereas N-acetyl-L-hydroxyproline loosens collagen structures. It was noted that incubation in ScaleS0 can greatly render a fixed sample adaptable to tissue clearing solutions. This adaptation process was achieved in the original ScaleA2 protocol by a freeze/thaw procedure that involved cryoprotection, OCT embedding, and re-fixation. These time-consuming and laborious steps in the original protocol can be replaced by a simple incubation in ScaleS0. Second, the permeable (adapted) sample is incubated sequentially in ScaleS1, ScaleS2, and ScaleS3. These urea-containing and salt-free ScaleS solutions gradually clear the sample. Finally, the sample is restored by simple washing with PBS(–) (deScaling) for ≥ 6 hrs, and then incubated in ScaleS4 for 12 hrs prior to observation. Fresh ScaleS4 is used as the mounting medium.

Although our original protocol using ScaleA2 specified the incubation temperature of 4 °C (Ref. 2), we found that the clearing processes were enhanced at higher temperatures. This temperature effect applies to ScaleS as well. In the new protocol, the incubation temperature is 37 °C for all ScaleS solutions but 4 °C for PBS(–).

The incubation occurs in a temperature-controlled orbital shaker (70–80 rpm/min). Occasionally, micelles may form in the mounting (ScaleS4) solution, and can be removed by decreasing the concentration of DMSO from 25% to 15%.

(c) AbScale protocol. The formulas of the AbScale solution and AbScale rinse solution are as follows.

[AbScale solution: a PBS(–) solution containing 0.33 M urea, 0.1–0.5% Triton X-100]

[AbScale rinse solution: a 0.1× PBS(–) solution containing 2.5% BSA, 0.05% (w/v) Tween-20].

The formulas of all other solutions are shown in Supplementary Table 1. The procedure assumes that an adult mouse cerebral hemisphere or a 1–2-mm-thick brain slice is processed as a sample to be immunolabeled/cleared. The method starts with the sequential incubation of a fixed brain sample in multiple solutions: ScaleS0, ScaleA2, ScaleB4(0), and ScaleA2 for permeabilization/clearing. Then, after deScaling by PBS(–) wash, the sample is incubated with a fluorescently labeled primary Ab (Direct IHC) or a primary Ab and then a fluorescently labeled secondary Ab (Indirect IHC) in an AbScale solution. Before re-fixation with 4% PFA, the sample is rinsed in an AbScale rinse solution. Finally, the immunostained sample is optically cleared by incubation in ScaleS4.

(d) ChemScale protocol. The formulas of all the solutions are shown in Supplementary Table 1. This procedure assumes that a mouse cerebral hemisphere or a 1–2-mm-thick brain slice is processed as a sample to be labeled/cleared. When combined with AbScale, fluorescent chemicals can be put in an AbScale solution containing a fluorescently labeled primary antibody.

(e) ScaleSQ(0) protocol. (f) ScaleSQ(5) protocol. These procedures assume that a 1–2-mm-thick brain slice is processed as a sample to be cleared. The only problem is that ScaleSQ solutions must be kept constantly at temperatures above 30 °C; otherwise, urea would precipitate due to its high concentration. The formulas of ScaleSQ(0) and ScaleSQ(5) solutions are shown in Supplementary Table 1.