Anti v5 tag primary antibody

For the confocal imaging of 293T cells, high precision microscope cover glasses (Marienfeld) were coated with poly-L-lysine hydrobromide (p6282, Sigma-Aldrich) according to the manufacturer’s protocol. Cells were seeded onto cover glasses in normal growth medium and fixed in 4% Formaldehyde solution (AppliChem) in PBS 1x after 24h of incubation. Permeabilization and blocking of samples was performed in blocking solution (10% FCS, 0.3% Saponin (47036, Sigma-Aldrich) in PBS 1x) for 1h rocking. Anti-V5 Tag primary antibody (Thermo Fischer Scientific, #46-0705) was diluted 1:500 in blocking solution and applied for 2h at room temperature, rocking. Samples were washed three times in blocking solution and anti-mouse Alexa Fluor 594 (Thermo Fischer Scientific, #A-11005) was applied 1:400 in blocking solution for 1h at room temperature, rocking. After three times washing in blocking solution nuclei were counterstained with DAPI 1:1000 in PBS 1x, for 10min, rocking. Cover glasses were mounted onto microscopy slides using ProLong Gold (Thermo Fischer Scientific) antifade mountant. Image acquisition was performed on a confocal laser scanning microscope (Zeiss LSM 780, Carl Zeiss AG), equipped with an Airyscan detector using ZEN black 2.3 (Carl Zeiss AG).