Deltavision spectris imaging system

Cells were fixed with 4% paraformaldehyde (PFA) for 8 min at room temperature (RT). After fixation, 0.1% Triton X-100 (Sigma-Aldrich) was applied for permeabilization. Cells were incubated with primary antibodies for 1 h at RT. Cells were incubated with Alexa Fluor 488- or 594-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen; 1:1000 dilution) for 1 h at RT. For staining actin filaments, Alexa Fluor 488- or 594-conjugated phalloidin (Invitrogen) was used according to the manufacturer’s protocol. For immunofluorescence staining of the mouse tumor, tumor tissues were fixed in 4% PFA overnight in 4 °C, dehydrated in 30% sucrose solution, and embedded in tissue freezing medium (Leica). Cryosections were blocked with 3% donkey serum in PBST (0.3% Triton X-100 in PBS) and then incubated at 4 °C overnight with primary antibodies. The samples were washed five times with PBS, followed by incubation with secondary antibodies (Invitrogen; 1:1000 dilution) for 2 h at 4 °C. Fluorescence images were acquired using a DeltaVision Spectris Imaging System (Applied Precision) and LSM800 confocal microscope (Carl Zeiss).

Live imaging of RPE1 cells stably expressing H2B-mCherry was performed with 35 mm petri dishes with a 14 mm glass-bottom microwell (MatTek) on a DeltaVision Spectris Imaging System (Applied Precision) equipped with an environmental chamber.