For immunostaining, neurons were first cooled to room temperature for 5 min. 250 μl of medium was removed per well, leaving approximately 220 μl, to which 32 μl of 32% paraformaldehyde (Electron Microscopy Sciences, #15714) was directly added, for an effective concentration of 4% paraformaldehyde (PFA). The PFA/neuron medium solution was gently mixed and incubated for 30 min at room temperature.
