Evos fl auto imaging system

Pictures were acquired with an inverted microscope EVOS^®® FL Auto Imaging System (Thermofisher, Waltham, MA, USA) and negative controls were used to adjust image acquisition parameters. ICC pictures were acquired with monochrome camera on DAPI (357/447 nm) fluorescence channel for Hoechst staining and GFP (470/525 nm) fluorescence channel for Alexa 488 staining.
Non fluorescent images were acquired with color brightfield image mode.

A total of 5000 cells grown on cover slips were washed with PBS and fixed with 4% formaldehyde. The cells were incubated with an anti-glut1 antibody (Cat #PA1-46152; Thermo Fisher Scientific) in PBS containing 5% FBS and 0.05% Triton for 30 min at 37°C. The cells were then washed four times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated antibodies for 30 min. The cells were then washed four times and images were obtained using the EVOS FL Auto Imaging System (Thermo Fisher Scientific). Images were used to quantify glucose transporter 1 (GLUT1) expression using MetaMorph (Molecular Devices, San Jose, CA, United States).

Tissue sections were deparaffinized in xylene followed by decreasing grades of ethanol. Samples were then quenched in 3% hydrogen peroxide and pretreated to promote antigen retrieval by using a steaming method with citrate buffer solution (pH 6.0). Slides were incubated in Dako Protein Block (Agilent), and then incubated with primary antibody to KRT18 (Abcam, ab181597) at a dilution of 1:2000 or Ki67 (Cell Signaling Technology, 12202S) at a dilution of 1:200. KRT18 antibody was validated using mouse liver as positive control, and Ki67 antibody was validated using mouse intestine as positive control. The samples were washed and incubated in Dako Rabbit Polymer (Agilent). Slides were further incubated with chromogen diaminobenzidine tetrahydrochloride (DAB). For amplification and visualization of our KRT18 or Ki67 signal, slides were counterstained with hematoxylin, rehydrated, and then mounted. For duct quantification, slides were imaged on an EVOS FL Auto Imaging System (ThermoFisher Scientific) and analyzed using ImageJ (NIH). All KRT18 or Ki67 positively stained ducts were detected using the Color Threshold tool in ImageJ. Based on KRT18 IHC images, ducts were quantified and sorted into two categories, either large or small ducts as defined by a cutoff of 1906 µm². This cutoff was derived from taking the arithmetic mean of all ducts detected in the vehicle-treated group.

For immunocytochemistry, glioblastoma stem-like cells were seeded at low density of 5000 cells per well (96-well plate). Cells were grown for 2–3 days on laminin and then fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. PFA was washed off with 1× PBS and then cells were permeabilised with PBS-0.1% Triton X-100 (PBS-T). Cells were stained for cytoskeletal structures to reveal cellular morphology. Cells were incubated with anti-nestin (sc-23927) at 1:100 titration for 1 h at room temperature with gentle agitation. After which, non-bound antibody was removed, cells were washed thrice with PBS-T, and then incubated with AlexaFlour488 conjugated secondary (anti-mouse) at 1:400 titration for 1 h at room temperature. Alternatively, cells were stained with ActinGreen^TM 488 ReadyProbes^TM, as per the manufacturer’s protocol. Cells were counterstained with 1:10,000 Hoechst 33342 (ThermoFisher). For imaging, an EVOS FL-auto imaging system (ThermoFisher) was used.

To evaluate the cytotoxicity of Ang II, the cells were resuspended in 400 µl of PBS, stained with propidium iodide (PI) (10 µg/ml) for 15 min at room temperature, and immediately analyzed by flow cytometer (BD FACSCanto II). An immunofluorescence imaging using the EVOS® FL Auto Imaging System (Thermo Fisher Scientific) was performed to confirm the flow cytometry experiment. The PI/Hoechst 33342 (10 µg/ml) combination staining was used to assess cell necrosis and apoptosis. After 15 min incubation with Hoechst at 37°C, the cells were centrifuged, and the pellets were resuspended in 300 µl PI (10 µg/ml) staining solution at room temperature for another 15 min. Immediately, the cells were analyzed by flow cytometry using (470/50 nm) emission light for Hoechst and (570/30 nm) for PI detection.