Tetramethylrhodamine conjugated phalloidin

NIH3T3 cells were seeded at 8000 cells/well in Millicell^® EZ 8-well chamber slides (Merck KGaA, Darmstadt, Germany) and transfected with 600 ng of each pTargeT^TM construct 24 h after seeding. Transfected cells were fixed with 4% paraformaldehyde at 48 h post-transfection for 20 min on ice, then permeabilized with 0.1% Triton X-100 in 1X PBS for 15 min at room temperature. After washing with 1X PBS, cells were blocked with 1% BSA in PBS for 20 min at room temperature, and then incubated in a 1:100 dilution of tetramethylrhodamine-conjugated phalloidin (Invitrogen; Thermo Fisher Scientific, Inc.) in 1X PBS for 1 h at room temperature with gentle shaking. The cells were again washed with 1X PBS before counterstaining the nuclei with Hoechst 33258 (1 µg/µL) for 5 min at room temperature. After the final washing step in 1X PBS, the cells were mounted in SlowFade^TM Diamond antifade mountant (Invitrogen; Thermo Fisher Scientific, Inc.) and were visualized under an inverted fluorescence microscope (IX83, Olympus Corporation), using a red fluorescent filter (λex/λem: 490/525 nm) to visualize filamentous actin structures, and a blue fluorescent filter (λex/λem: 355/465 nm) to visualize the nuclei.

Using Millicell^® EZ 8-well chamber slides (Merck KGAa, Darmstadt, Germany), NIH3T3 cells were seeded at 6500 cells/well and transfected with 300 ng of each pTargeT^TM construct 24 h after seeding. After 48 h, transfected cells were fixed with 4% paraformaldehyde for 20 min on ice, then permeabilized with 0.1% Triton X-100 in 1X PBS for 15 min at room temperature, and washed thereafter with 1X PBS. The cells were then blocked with 1% BSA in PBS for 20 min at room temperature, and incubated in a 1:100 dilution of tetramethylrhodamine-conjugated phalloidin (Invitrogen; Thermo Fisher Scientific, Inc.) in 1X PBS for 1 h with gentle shaking at room temperature. After another wash with 1X PBS, nuclei were counterstained with Hoechst 33258 (1 µg/µL) for 5 min at room temperature. After the final washing step in 1X PBS, cells were mounted using SlowFade^TM Diamond antifade mountant (Invitrogen; Thermo Fisher Scientific, Inc.) and visualized under a fluorescence microscope (IX83, Olympus Corporation). A red fluorescent filter (λex/λem: 490/525 nm) was used to visualize filamentous actin structures, and a blue fluorescent filter (λex/λem: 355/465 nm) to visualize the nuclei.