HCC1937, HCC1937 BRCA1, and CAL51 cells were cultured alone or transfected with pCDNA3, BRCA1a, BRCA1a Mut#1, for 24 hours in six-well plates onto glass cover slips overnight. The cells were washed and fixed in icy methanol for 5 minutes, and blocked using 10% BSA for 60 minutes, followed by primary polyclonal Rabbit anti-collagen antibody 1:250, primary polyclonal anti-fibronectin and β-catenin antibody (Santa Cruz), 1:500 diluted in 1.5% BSA made in PBS at 25°C for 1 hour and Alexa 488 goat anti-Rabbit/Alexa 568 goat anti-mouse (Molecular Probes) diluted in 1.5% BSA/PBS for 50 minutes and stained (Hoechst 33258, Pentahydrate, Life technologies). The cover slips were mounted with Vectashield mounting medium for fluorescence (H-1000 from Vector). The stained cells were examined by LSM 700 Confocal Microscope, equipped with 63X oil Ph immersion objectives. Composite filter cubes were used for the 488–405 as described previously [25 (link)].
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