14 mm coverslip bottom dishes

Cells were grown on Matrigel-coated 8 mm diameter circular glass coverslips, on 14 mm coverslip bottom dishes (Mattek Corporation) or on eight-well µ-Slide (ibidi). Cultures were fixed for 10 min in 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature. Coverslips were then washed three times with PBS followed by 15 min permeabilization with 0.2% Triton X-100 in PBS. Subsequently the coverslips were washed three times in wash buffer (0.05% Tween-20 in PBS) for 5 min and blocked for 20 min in 200 ml of wash buffer plus 5% normal donkey serum (Jackson ImmunoResearch Laboratories). The primary antibodies were diluted in 5% normal donkey serum in PBS and incubated with the tissues for 1 h at room temperature followed by three washes in wash buffer for 5 min each. The secondary antibodies were diluted in 5% normal donkey serum in PBS and incubated with the tissues for 1 h at room temperature followed by three washes in wash buffer for 5 min each. Afterwards, the 8 mm coverslips were mounted on microscopy glass slides using ProLong Glass Antifade (Life Technologies), cured overnight at room temperature and then sealed with clear nail polish. Coverslips in coverslip bottom dishes or µ-slides were stored in PBS at 4 °C for 3D imaging.