Patient derived neurons were washed with PBS and fixed in fresh 4% paraformaldehyde in PBS overnight rocking at 4°C. Cells were washed with PBT and blocked in PBT + BSA for 1 h at room temperature. Primary antibody was added to PBT overnight rocking at 4°C. Antibodies: 1:100 Syt-1, Vesicular Glutamate 1 and 2 (VGlut1/2, Synaptic Systems #135503) or Postsynaptic Density 95 (PSD95, Abcam #ab18258), Acetylated-Tubulin (Ac-Tubulin, Abcam #ab179513). Primary antibody was washed off three times in PBT and incubated in secondary antibody shaking overnight at 4°C (1:500 488- 555- 680- (Jackson ImmunoResearch, see above) in PBT + BSA). Secondary was washed off three times in PBT, rocking for 2 h at room temperature. Cells were washed three times in PBT and mounted on slides with DAPI (Prolong Gold Antifade with DAPI (Life Technologies #P36935). Imaging was performed on an inverted Zeiss LSM700 Laser Scanning Confocal and processed on Fiji (ImageJ).
SH-SY5Y cells were plated on coverslips and differentiated over 4 days with retinoic acid medium. The same imaging protocol was performed as above. Antibody: 1:200 Beta-Actin.
SH-SY5Y cells were plated on coverslips and differentiated over 4 days with retinoic acid medium. The same imaging protocol was performed as above. Antibody: 1:200 Beta-Actin.