Imager 1.0 apotome microscope

Spheroids were prepared and processed as previously described^{68 (link)}. Briefly, HT-29 cells (1500 cells/well) were seeded in Ultra-Low Attachment 96-well plates (Corning) in DMEM supplemented with 10% heat-inactivated FBS, 10 mM D-glucose and 2 mM L-glutamine. For immunohistochemical studies, spheroids were incubated for 24 h with 2 µM Alexa Fluor 568-conjugated pH-low insertion peptide variant 3 (pHLIP V3; NH₂-ACDDQNPWRAYLDLLFPTDTLLLDLLW-COOH^{69 (link)}) to label acidic regions. Spheroids were then washed twice in PBS, fixed in 4% PFA, harvested and embedded in OCT. Frozen sections (5 µm) were stained with either BODIPY^TM 493/503 (#D3922; Thermo Fisher Scientific) or anti-Ki67 antibody (#556003, BD Biosciences). Sections were incubated with Alexa Fluor 568-conjugated anti-mouse secondary antibodies (#A11031; Thermo Fisher Scientific), and nuclei were counterstained with DAPI. Slides were prepared with fluorescence mounting medium (Dako), and staining was visualized with a Zeiss Imager 1.0 Apotome microscope. All spheroid samples from a same experiment were imaged by using the same gain and exposure settings.

Cells grown on coverslips were fixed with 4% PFA for 10 min. After blocking with 5% BSA for 1 h, cells were incubated overnight at 4 °C with anti-E-cadherin and anti-vimentin antibodies (#14472 and #5741, respectively; Cell Signaling Technology). Cells were then incubated with Alexa Fluor 488- and 568-conjugated anti-rabbit secondary antibodies (#A11034 and #A11036, respectively; Thermo Fisher Scientific) for 1 h and nuclei were counterstained with DAPI. For neutral lipid staining, PFA-fixed cells were incubated with 0.5 µg/ml BODIPY 493/503 (#D3922; Thermo Fisher Scientific) for 30 min at room temperature. Slides were prepared with a fluorescence mounting medium (Dako) and staining was visualized with a Zeiss Imager 1.0 Apotome microscope.