Alexa fluor 555

To prepare the paraffin-embedded histology sections, samples were decalcified with 10% EDTA for 3 weeks at room temperature after image collection by micro-CT. Sections were stained with H&E for morphological analysis.
EdU-incorporated proliferative cells were detected using the Click-iT EdU Cell Proliferation Kit for Imaging Alexa Fluor 488 dye (Invitrogen) according to manufacturer instructions. Following the detection of EdU-labeled cells, molecular markers were co-labeled by immunohistochemistry, as previously described^{40 (link)}. The primary antibodies used were as follows: mCherry (1:2,000; M11217; Invitrogen), Gli1 (1:400; 78259; NOVUS, Littleton, CO, USA), Ring1b (1:100; 101273; Abcam, Cambridge, UK), Cbfa1/Runx2 (1:400; 035; MBL Life Sciences, Nagoya, Japan), Sp7/Osterix (1:800; 22552; Abcam), and PCNA (1:200; 2586; Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were as follows: goat anti-rabbit IgG antibody Alexa Fluor 555 (1:400; Invitrogen) and goat anti-mouse IgG antibody Alexa Fluor 555 (1:400; Invitrogen). For the detection of Gli1, Alexa Fluor 555 Tyramide SuperBoost Kit goat anti-rabbit IgG (Invitrogen) was used to amplify the immunoreactivity according to manufacturer instructions. The samples were enclosed with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).

The primary antibodies used were: rabbit anti-XB130 (1:1,000, Abgent, San Diego, CA), rabbit anti-Ki67 (1:100, Lab Vision, Fremont, CA), rat anti-Ly-6B.2 (1:10,000, AbD Serotec, Raleigh, GC), rat anti-F4/80 (1:500, AbD Serotec), goat anti-podoplanin (PDPN) (1:50, R & D Systems, Minneapolis, MN), rabbit anti-surfactant protein C (SFTPC, for type II cells)(1:1,000, Seven Hills Bioreagents, Cincinnati, OH) and rabbit anti-phospho-GSK-3β-Ser9 (1:500, Cell Signaling, Beverly, MA). After incubation with primary antibodies, sections were incubated with appropriate secondary antibodies. IHC was performed using a Vectastain ABC kit (Vector Laboratories, Burlington, Canada) with 3-3-diaminobenzidine as chromogen, and sections were counterstained with hematoxylin, and images were captured via Olympus BX51-FL. For IF, the secondary antibodies used were: donkey anti-goat Alexa Fluor^® 488, donkey anti-rabbit Alexa Fluor^® 555 and goat anti-mouse Alexa Fluor^® 555 (1:200, Invitrogen, Burlington, Canada), and sections were mounted with Prolong Gold Antifade Mountant with DAPI^® (Invitrogen). The slides were examined with an Olympus BX-51, and images were captured via QImaging colour camera (Olympus Co, Ltd). We randomly chose 5 fields (×200) per slide for positive cell counting.

For the preparation of ovarian paraffin blocks, ovaries were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C. Ovaries were dehydrated and embedded in paraffin. For histological analysis, 5-µm-thick paraffin sections were deparaffinized and stained with hematoxylin and eosin. For immunostaining, ovarian paraffin sections were deparaffinized and autoclaved in target retrieval solution (DAKO) for 10 min at 121 °C. Sections were blocked with Blocking One Histo (Nacalai) for 1 h at room temperature and then incubated with primary antibodies (anti-H2AK119ub, anti-DDX4, and anti-SYCP3 at 1:200 dilution) overnight at 4 °C. Localization of the primary antibody was performed by incubation of the sections with the corresponding secondary antibodies (Donkey Anti-Mouse IgG (H + L) Alexa Fluor 488, A-21202; Donkey Anti-Rabbit IgG (H + L) Alexa Fluor 555, A-31572; Donkey Anti-Rabbit IgG (H + L) Alexa Fluor 488, A-21206; Donkey Anti-Mouse IgG (H + L) Alexa Fluor 555, A-31570; Invitrogen) at 1:500 dilution for 1 h at room temperature. Finally, sections were counterstained with DAPI and mounted using 20 mL undiluted ProLong Gold Antifade Mountant (ThermoFisher Scientific, P36930). Images were obtained by confocal laser scanning microscope (A1R, Nikon) and processed with NIS-Elements (Nikon).

2D HCT116 and HT29 cells were grown (8 × 10⁴ cells/well) in eight-well chambered coverslips (Ibidi) and treated after 2 days of adherence for 48 h with increasing concentrations of SARB (10, 20, 50 µM). Spheroids (3D) at day 21 derived from SFA and 2D cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at RT, permeabilized with 0.1% TritonX in PBS (15 min at RT), blocked for 30 min with 3% BSA (w/v) in PBS, and stained with a 1:250 dilution of primary antibody against CD133 (Miltenyi Biotec) or 1:100 of ß-Catenin (D10A8) XP (Cell Signaling) overnight at 4 °C. The next day, after a washing step, the spheroids stained with CD133 were incubated with a 1:250 goat anti-mouse dilution of the secondary antibody (Alexa Fluor 555, Invitrogen), where the cells labeled with ß-Catenin were incubated with 1:100 goat anti-rabbit IgG (Alexa Fluor 555, Invitrogen) for 1 h RT, washed again, stained with 1:1000 Hoechst 33342 (Sigma) in PBS (1 mg/ml stock solution) per well and imaged with a Nikon Eclipse Ti–S.