The following antibodies were used in this study: primary antibody—GFP (1:500 dilution; AB3080, Millipore); secondary antibodies—Cy3 AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H+L) (1:500 dilution; 111-165-003, Jackson ImmunoResearch), Cy5 AffiniPure goat anti-rabbit IgG (H+L) (1:500 dilution; 111-175-144, Jackson ImmunoResearch), and Alexa Fluor 594 goat anti-rabbit IgG (H+L) (1:500 dilution; A-11037, Life Technologies).

For immunostaining, 1-mm-thick Thy1-GFP-M brain slices were immunostained for GFP using the iDISCO protocol (25). For pretreatment with methanol, brain slices were washed twice with PBS for 1 hour and then placed in 50 and 80% methanol (in PBS) for 1 hour at each step and twice in 100% methanol for 1 hour. The sections were bleached with 5% H2O2 in 20% dimethyl sulfoxide (DMSO) (472301, Sigma-Aldrich)/methanol at 4°C overnight. After bleaching, the slices were washed with methanol for 1 hour twice, followed by 20% DMSO/methanol for 1 hour twice, 80 and 50% methanol for 1 hour at each step, PBS for 1 hour twice, and finally PBS/0.2% Triton X-100 (T8787, Sigma-Aldrich) (PBST) for 1 hour twice. For the immunostaining step, the slices were incubated in PBS/0.2% Triton X-100/20% DMSO/0.3 M glycine (50046, Sigma-Aldrich) at 37°C overnight, blocked in PBS/0.2% Triton X-100/10% DMSO/6% goat serum at 37°C for 1 day, washed in PBS/0.2% Tween 20 (P2287, Sigma-Aldrich) with heparin (10 mg/ml; PTwH) overnight, and finally incubated with primary antibody dilutions in PTwH/5% DMSO/3% goat serum at 37°C with slight shaking on an oscillator for 2 days. The slices were then washed for 1 day and incubated with secondary antibodies diluted in PTwH/3% goat serum at 37°C with slight shaking on an oscillator for 2 days. The slices were finally washed in PTwH for 2 days prior to imaging.

For nuclei staining, 1-mm-thick Thy1-GFP-M brain slices were incubated with PI (2 μg/ml; P1304MP, Life Technologies) in PBS/0.2% Triton X-100 for 1 day at room temperature with gentle oscillation and then washed with PBS.

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