Rabbit anti synaptophysin

An MEA plate was fixed on the day of experiment (Table 1, Plate 8) with 4% paraformaldehyde (PFA, pH~6.9) and washed gently with phosphate buffer saline (PBS, Sigma). Blocking and permeabilization of the cells were performed using the Blocking Solution. Afterwards, a second wash was performed, and preparation was incubated with primary antibodies in the Dilution Solution, overnight at 4°C. Utilized primary antibodies were chicken Anti β-III Tubulin (Abcam) and rabbit Anti-Synaptophysin (Santa Cruz) with 1:200 and 1:50 dilutions, respectively. After that, the primary antibodies were washed out with PBS and secondary antibodies were added and incubated for three hours at room temperature. Secondary antibodies were Alexa Fluor 633 goat anti-chicken Immuno-globulin-G (IgG) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) with 1:100 and 1:400 dilutions, respectively. DAPI (Invitrogen) was added to the sample at 1 μg/ml concentration. A final wash with 2:1000 PBS-Tween20 solution was performed in dimmed light and preparation was kept in PBS-Azide with 1:1000 dilution.