Millicell ez 8 well chamber slides

Cells were cultured in Millicell EZ 8-well chamber slides (Merck Millipore, Watford, UK). To fix the cells, the culture medium was removed and the cells were washed with PBS and then fixed in 100% ice-cold ethanol. To proceed with immunofluorescence staining, the cells were washed 3 times in PBS and then permeabilised using 0.1% Triton X-100 (Sigma-Aldrich, Dorset, UK). Blocking buffer (5–10% donkey serum (D9663, Sigma-Aldrich, Dorset, UK)) in PBS was added to each well. The slide was left for 3 h at room temperature in a blocking buffer. The chamber slide was then incubated with primary antibodies for an hour on the bench or at 4 °C overnight. Secondary antibodies were (1:500) with the addition of DAPI (1:100), and each primary antibody was incubated with the corresponding secondary for a further hour. Following three washes in PBS, the slides were mounted in FluorSave™ (Calbiochem, Nottingham, UK) and allowed to dry before being visualised using an Olympus BX51 microscope with a Hamamatsu Orca ER digital camera at ×40. Images were analysed using ImageJ.

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.

For immunofluorescence staining, cells were cultured in Millicell EZ 8-well chamber slides (Merck Millipore, Watford, UK) for 18 h at a seeding density of 5 × 10⁴ cells per well. Cells were fixed in 500 μl of ice cold 100% ethanol at −20 °C. Cells were permeabilised with 0.1% Triton X 100 (Sigma Aldrich). Primary antibodies (diluted to 1:100) and secondary antibodies (1:500 for Alexa secondary antibodies and 1:1000 for DAPI) were prepared in blocking buffer in blocking buffer and applied. Slides were washed and mounted using FluorSave (Calbiochem, Nottingham, UK) and visualised using an Olympus BX51 microscope with a Hamamatsu Orca ER digital camera at × 40. Integrated density was measured using ImageJ.