Immunofluorescence Staining of Cell Cultures

Cells cultured on glass cover slips were fixed with 3% formaldehyde, permeabilized with 0.05% Triton-X and blocked with phosphate-buffered saline (PBS) containing 10% goat serum and 1% BSA. Cover slips were incubated overnight with primary antibody dilutions (1:250) prepared in 0.2% BSA in PBS 1 ×, and subsequently washed and incubated with an Alexa Fluor-488 goat anti-mouse antibody (Invitrogen, Waltham, MA, USA) for 1 h at room temperature. Slides were then exposed to DAPI (1 μg/ml) in PBS at room temperature for 5 min. Cover slips were mounted using VECTASHIELD with DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured utilizing a Leica Fluorescent microscope. IHC analysis was carried out as previously described;^{61 (link)} antibodies used were anti-E-cadherin, anti-fibronectin (GeneTex, Irvine, CA, USA) and anti-p27 (Cell Signaling, Danvers, MA, USA).