Roti mountfluorcare mounting media

5x10⁴ LLC1 cells were plated in 24 well plates on glass cover slips in DMEM or embedded into 0.5 mg/ml lr-ECM/DMEM mixture under 2D or 3D cell culture conditions, respectively. Following 48 h of growth, cells were washed twice with PBS and fixed for 10 min. with 4 % PFA (Carl ROTH, Germany) solution in PBS at room temperature. Cell permeabilization was performed with ice-cold 0,1 % Triton X-100 in PBS for 10 min. Staining was accomplished with Alexa®633 Phalloidin (ThermoFisher Scientific, USA) in PBS containing 1 % BSA for 30 min and 5 μg/ml Dapi (Sigma, USA) in PBS for 3 min at room temperature. All staining steps were followed by 3 wash steps in PBS for 5 min at room temperature. Finally, slides were mounted with Roti®-MountFluorCare mounting media (Carl ROTH, Germany). Images were obtained using Zeiss LSM 7 Duo Live confocal microscope (Zeiss, Germany) and 40x/1.3 immersion objective and excitation wavelengths of 405 nm and 633 nm.

Cells were seeded onto coverslips (Ø 13 mm) coated with 5 µg/cm² rat collagen (ibidi, Gräfelfing Germany) for the fluorescence microscopy experiments. Following the cultivation procedure described above, the cells were washed twice in PBS, fixed for 20 min with 4% paraformaldehyde and washed twice again with PBS. Permeabilization was accomplished by 10 min incubation with 0.1% Triton^® X 100 (Roth, Karlsruhe, Germany) in PBS and another two washing steps. The blocking of unspecific binding sites was achieved by the application of 5% bovine serum albumin in PBS for 1 h. Cells were washed twice again with PBS. A primary antibody dilution in 1% bovine serum albumin in PBS was applied (ab137443, 1:100; Abcam, Cambridge, UK) for 2 h. Subsequently, another double washing step was completed before the cells were incubated for 1 h in the presence of a secondary antibody dilution in 1% BSA in PBS (ab150078, 1:400; Abcam, Cambridge, UK). After washing twice, the cells were counterstained with a 0.25 µM DAPI solution (Abcam, Cambridge, UK). After a final double wash step, the coverslips were mounted in ROTI^® Mount FluorCare mounting media (Roth, Karlsruhe, Germany). A BZ-X810 microscope (Keyence, Osaka, Japan) was used to capture fluorescence images.