Immunostaining Neurons for Stau2 and VGLUT1

For immunostaining^{28 (link)} neurons were washed with warm Hanks′ Balanced Salt Solution (HBSS) and then fixed with warm 4% PFA in HBSS for 10 min. Fixed cells were washed with HBSS and permeabilized with 0.1% Triton X-100 in HBSS for 5 min. The following antibodies were used: (i) polyclonal antibodies, i.e. selfmade rabbit anti-Stau2^{53 (link)}, guinea pig anti-VGLUT1 (Synaptic Systems, 419005); (ii) secondary antibodies, i.e. donkey anti-rabbit and goat anti-guinea pig Alexa555 or Alexa647 conjugated (Life Technologies, A31572, A31573, A21450). Coverslips were mounted on slides with Prolong antifade mounting medium (Invitrogen).