Anti gfp d5.1 rabbit monoclonal antibody

0:5×10⁵ cells were seeded on poly-Lysine coated high-precision glass coverslips (18 mm round, #1.5) in 12-well culture plates one day prior to transfection. Transfection was performed as described for flow cytometry experiments. A total amount of 200 ng DNA (20 ng synTFs and 180 ng ssDNA) was used for transfection experiments. PEI was scaled to 12-well plate volume of 100 μL total transfection mix. 48 h post transfection, cells were washed with 1x PBS, fixed with 2% PFA (Fisher Scientific) and blocked for 30 min with 10% Goat serum (VWR) in 1x PBS after washing. Immunodetection was performed using anti-HA-tag (6E2) mouse monoclonal antibody (Cell Signaling) and anti-GFP (D5.1) rabbit monoclonal antibody (Cell Signaling) 1:200 in 1%BSA/PBS overnight. Cells were washed with 0.1% Triton X-100 and incubated with anti-mouse IgG Alexa Fluor 488 (#4408, Cell Signaling) and anti-rabbit IgG Alexa Fluor 647 antibodies 1:1000 in 1%BSA/PBS for 1 h. After washing with 0.1% Triton X-100, nuclei were stained with 2 μg/mL Hoechst-33342 (Thermo Fisher Scientific) and mounted on glass slides using Prolong Gold Antifade (Thermo Fisher Scientific). Image acquisition was performed at least 16 h after mounting coverslips on glass slides.