Following aspiration of the PFA/medium mix, ice-cold blocking/ permeabilization buffer (PBS containing Ca2+ and Mg2+ ThermoFisher Scientific (21300-058) supplemented with 0.1% Triton-X (Sigma, #T8787) and 5% Donkey serum (Jackson, #017-000-121) was gently added. Neurons were incubated with blocking/permeabilization buffer on ice for 30 min, followed by an additional 30 min at room temperature. It is critical to perform this step on ice to retain neurite morphology during permeabilization and to prevent fragmentation of neurites.
