Vectashield medium

Upon treatment the cells were immunostained for the following Extracellular matrix protein Fibronectin using the antibody obtained from “The Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242”: 1- Fibronectin, cell binding domain - P1H11 was deposited to the DSHB by Wayner, E.A. (DSHB Hybridoma Product P1H11). For this assay, the cells were fixed in 4% paraformaldehyde in PBS for 5 min. Permeabilization was performed with 0.3% Triton X-100 in PBS, followed by blockage with 5% BSA (Bovine Serum Albumin; Sigma Aldrich) in PBS for 1 h at room temperature. After blocking, washes were performed using 1x PBS for 3 times/10 min followed by incubation with the primary antibody mentioned above at a concentration of 0.3 μg/ml overnight. Upon PBS washes cells were incubated with secondary antibody, anti-mouse Alexa 488 (1:400, Molecular Probes), followed by actin staining with Phalloidin 546 (1:400, Molecular Probes) and DAPI (1:400), for 2 h at room temperature protected from light. The coverslips were washed with 1x PBS, 3 times/10 min, mounted in VectaShield medium (Invitrogen) and analyzed under confocal microscopy (Leica TCS SP5 AOBS).