For μLP perfusions, we used or replaced medium with either Hibernate A (BrainBits, LLC) containing B27 (1:50; Invitrogen) or HBS. To visualize perfusions, we added 1-2 μM of the low molecular weight Alexa Fluor 488 hydrazide (570 m.w.; A10436; Invitrogen) or Alexa Fluor 568 hydrazide (731 m.w.; A10437; Invitrogen). Alternatively, to monitor perfusion without added fluorescent dyes (in the case of DHPG experiments), we first perfused medium containing 2.5-5% BSA, then added perfusate without BSA to center channel, which allowed us to visualize the start and duration of the perfusion due to the mismatch in refractive indices of the solutions without the use of a fluorescence microscope (see Figure 8A). To show isolation of pre- and post-synaptic compartments, we also used Alexa Fluor 633 hydrazide (1150 m.w.; A30634; Invitrogen).
