For immunofluorescence analysis cells were grown in 8-well chamber slides (BD Biosciences) in 3D culture conditions (BD Biosciences). Cells were fixed in 4% formalin for 20 min and washed with PBS-glycine (0.7%) before blocking with PBS/0.1% Bovine Serum Albumin (BSA)/0.2% Triton X-100/0.05% Tween-20/10% goat serum for 1.5 hr.
Cultured MCF10A 3D acini were incubated for 2 hr with antibodies to Ki-67 (clone MM1) diluted 1:50 or to integrin-alpha6 (clone GoH3) diluted 1:100 in blocking buffer prior to incubation with Alexa Fluor® 488 Goat Anti-Mouse or Donkey Anti-Rat secondary antibodies, respectively, for 1 hr. All incubation steps were carried out at room temperature. Counterstaining with DAPI was then followed by mounting using the ProLong Antifade agent (ThermoFisher Scientific).
Indirect TUNEL was performed using The ApopTag® Red In Situ Apoptosis Detection Kit (Merck Millipore) following the manufacturer’s protocol. Slides were analyzed on a Zeiss LSM 710 confocal microscope using a 20X objective.
Cultured MCF10A 3D acini were incubated for 2 hr with antibodies to Ki-67 (clone MM1) diluted 1:50 or to integrin-alpha6 (clone GoH3) diluted 1:100 in blocking buffer prior to incubation with Alexa Fluor® 488 Goat Anti-Mouse or Donkey Anti-Rat secondary antibodies, respectively, for 1 hr. All incubation steps were carried out at room temperature. Counterstaining with DAPI was then followed by mounting using the ProLong Antifade agent (ThermoFisher Scientific).
Indirect TUNEL was performed using The ApopTag® Red In Situ Apoptosis Detection Kit (Merck Millipore) following the manufacturer’s protocol. Slides were analyzed on a Zeiss LSM 710 confocal microscope using a 20X objective.