Prolong gold antifade mountant containing dapi

Samples were collected from culture at day 30 and fixed in 4% (v/v) paraformaldehyde (PFA)/4% (v/v) sucrose for 20 min. Spheroids were dehydrated with 30% (w/v) sucrose overnight, frozen at –80°C in Tissue-Tek O.C.T. (Sakura, Alphen aan den Rijn, Netherlands) and sectioned at 10 μm thickness using a cryomicrotome (Cryostat I, Leica). Immunofluorescence was performed according to previously published methods (Rebelo et al., 2015 (link); Simão et al., 2015 (link)). Briefly, cells were permeabilized with 0.1% Triton X-100 (w/v) and blocked in 1% bovine serum albumin (BSA). Primary antibodies were diluted in 1% (w/v) BSA and incubated overnight at 4°C. Secondary antibodies were diluted in 1% (w/v) BSA and incubated for 1 h at RT. Samples were mounted in ProLong Gold Antifade Mountant containing DAPI (Thermo Fisher) and visualized using a fluorescence microscope (DMI6000B, Leica Microsystems). The primary antibodies used were anti-CD31, von Willebrand factor (vWF), collagen types I and IV, and fibronectin (all from Abcam, Cambridge, United Kingdom).

Tissues were fixed with 4% formaldehyde, embedded in paraffin blocks and thin sections (4 μm) were prepared. For hematoxylin and eosin staining, sections were stained as previously described^{29 (link)}. For collagen staining, sections were stained using Trichrome stain kit (Sigma) according to the manufacturer’s protocol. Images were acquired using ICC50HD camera (Leica).
Unfixed tissues were embedded frozen in optimum cutting temperature compound (Tissue-Tek) and cryostat sections (9 μm) were prepared on glass slides. For neutral lipid staining, sections were incubated with Oil Red O (Matheson Coleman & Bell) in 60% isopropyl alcohol for 30 min. Slides were then washed with distilled water and mounted using glycerol jelly. Images were acquired using ICC50HD camera (Leica). For immunohistochemistry, frozen sections were fixed with 100% acetone for 10 mins and blocked with 3% bovine serum albumin in phosphate buffer for 1 hr at room temperature. Sections were then incubated with rat monoclonal anti-CD45 (BioRad MCA1031G, dilution 1:200) overnight at 4 °C. After washing, sections were incubated with goat anti-rat IgG, Texas Red (Invitrogen T-6392, dilution 1:200) and mounted with Prolong^® Gold antifade mountant containing DAPI (ThermoFisher). Images were acquired using a Meta 510 confocal scope (Zeiss).

A549 cells were seeded (1 × 10⁴) on 8-well chamber slides (Corning, 354630). A day after cells were fixed by using 4% formaldehyde (Thermo Fisher Scientific, 28906) for 15 min at RT, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA (Cell Signaling Technology, 9998) for 1 h. DyLight™ 554 phalloidin (Cell Signaling Technology, 13054) was used to stain F-actin (1:200 dilution in PBS) for 15 min at RT. Chamber slides then incubated with Prolong Gold Antifade Mountant containing DAPI (Thermo Fisher Scientific, P36931).