MCF-10A cells were cultured in 3-D using Matrigel, as previously described [11 (link), 44 (link)]. Cell cultures in chamber slides were fixed with 4% paraformaldehyde, followed by staining for immunofluorescence using anti-ezrin antibody (3C12) from Santa Cruz Biotechnology (Santa Cruz, CA), Alexa 488-conjugated anti-mouse IgG and Hoechst 33342 from Life Technologies, Grand Island, NY.
For immunofluorescence using antibodies against the EMT markers and Ki67, MCF10A cells were densely plated on poly-lysine coated coverslips. After 14-day culture in minimal medium, cells were fixed and incubated with relevant primary antibodies and secondary fluorescein-conjugated horse anti-mouse IgG antibody (#FI-2000, Vector Laboratories, Burlingame, CA) or Texas Red®-conjugated goat anti-rabbit IgG antibody (#TI-1000, Vector Laboratories). The slides were mounted with Vectashield Mounting Medium plus DAPI (#H-1200, Vector Labs)
Samples were then photographed using Nikon C2 Confocal Microscope or Zeiss Axiovert 200M Microscope. For quantification of the size of acini or “islands”, the images in10x magnification were acquired by TissueFAXS 200 (Tissuegnostics, Vienna, Austria) or by Zeiss Axiovert 200M, followed by analysis using the ImageJ software.
For immunofluorescence using antibodies against the EMT markers and Ki67, MCF10A cells were densely plated on poly-lysine coated coverslips. After 14-day culture in minimal medium, cells were fixed and incubated with relevant primary antibodies and secondary fluorescein-conjugated horse anti-mouse IgG antibody (#FI-2000, Vector Laboratories, Burlingame, CA) or Texas Red®-conjugated goat anti-rabbit IgG antibody (#TI-1000, Vector Laboratories). The slides were mounted with Vectashield Mounting Medium plus DAPI (#H-1200, Vector Labs)
Samples were then photographed using Nikon C2 Confocal Microscope or Zeiss Axiovert 200M Microscope. For quantification of the size of acini or “islands”, the images in10x magnification were acquired by TissueFAXS 200 (Tissuegnostics, Vienna, Austria) or by Zeiss Axiovert 200M, followed by analysis using the ImageJ software.
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