Anti shank3

Cell culture media was from Invitrogen. Poly-L-lysine was from Sigma. The following primary antibodies were used: mouse monoclonal anti-Bassoon clone SAP7F407 (Abcam, UK), mouse monoclonal anti-Synapsin IIa (BD Biosciences, UK), rabbit polyclonal anti-Homer, rabbit polyclonal anti-Shank3, rabbit polyclonal anti-vGlut1 (Synaptic Systems, Germany). Secondary antibodies were from Jackson Immunoresearch (USA). APV, NBQX, TTX were from Tocris (UK).

For immunofluorescence analysis, DIV14-21 neurons in the microfluidic device were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature (RT). The fixation buffer was rinsed out with PBS, after which neurons were permeabilized with 0.2% Triton X-100 for 30 min and subsequently incubated for 30 min with a blocking solution containing 5% bovine serum albumin (BSA). Anti-Tau (Merck Millipore), anti-MAP2 (Merck Millipore), anti-synapsin (Synaptic Systems), and anti-Shank3 (Synaptic Systems) antibodies were applied to microchannels and the microfluidic device was incubated overnight at 4 °C. After incubating with primary antibodies, neurons were washed with PBS for 30 min and subsequently incubated with Alexa 488- and/or Alexa 546-conjugated secondary antibodies (Invitrogen) for 1 h, providing different color combinations as needed.