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Blocking is separated as in vivo and in vitro

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After washing 3 times with PBS, neurons were incubated with 5.5 M pHluorin-labelled Syt1 nanobody in the presence of either 30mM KCl or 1M TTX (Alomone labs) to stimulate neurons or to silence them, respectively, for a total of 20 min at 37 C in a humid chamber