Black line : Positive blank control (Molt4); Negative blank control (A549）
Green line : Primary Antibody (Rabbit Anti- SV2A antibody (SL487R) )
Orange line：Isotype Control Antibody (Rabbit IgG) .
Blue line : Secondary Antibody (Goat anti-rabbit IgG-AF488)/(Goat anti-rabbit IgG-AF647)
Molt4（Positive）and A549（Negative control）cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with SV2A Antibody(SL487R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Blank control:Molt-4.
Primary Antibody (green line): Rabbit Anti-SV2A antibody (SL487)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 20% PBST for 20 min at-20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
