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Pretreatment before staining was as follows: the fixed brains were washed two times in PBS for 1 h, incubated in 50% methanol (in PBS) for 1 h, 80% methanol for 1 h and two times in 100% methanol for 1 h. The samples were bleached with 5% H2O2 in 20% DMSO/methanol (one vol 30% H2O2/one vol DMSO/four vol methanol) at 4 °C overnight. Subsequently, samples were transferred to methanol for 1 h twice, then in 20% DMSO/methanol for 1 h twice, then in 80% methanol for 1 h, 50% methanol for 1 h, twice in PBS for 1 h, and finally in PBS/0.2% Triton X-100 for 1 h twice before further staining procedures. Pretreated samples were incubated in PBS/0.2% Triton X-100/20% DMSO/0.3 M glycine at 37 °C overnight, then blocked in PBS/0.2% Triton X-100/10% DMSO/6% donkey serum at 37 °C overnight. This was followed by a wash in PBS/0.2% Tween-20 with 10 μg ml−1 heparin (PTwH) for 1 h twice, then incubated in rat anti-mCherry (for tdTomato; Thermo Fisher Scientific, M11217; 1:500 dilution) in PTwH/5% DMSO/3% donkey serum at 37 °C for 8 d. After a 1-d long wash in PTwH, the samples were incubated in anti-rat Alexa Fluor 594 (Thermo Fisher Scientific, A21207; 1:300 dilution) in PTwH/3% donkey serum at 37 °C for 4 d. Samples were finally washed in PTwH before starting the clearing protocol, followed by LSFM imaging.

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