P6744

To label collagen deposition (indicative of fibrosis), 5 µm paraffin embedded tissue sections were stained with picrosirius red (365548 and P6744, Sigma) and 0.1% Fast Green (F7258, Sigma) at room temperature for 30 minutes. The relative quantification of fibrosis was measured using ImageJ. Ten 4× fields were imaged for each liver, representing a 2.95×2.21 mm area. Using the color threshold tool, red staining was isolated. The image is then converted to grey scale, and threshold is used to subtract remaining background. Measurement of the remaining area yielded the area of the slide with red staining. This was normalized to the area of the tissue (vessel lumen subtracted) for each slide to yield the percent of tissue with picrosirius red staining. Data was processed by macro to remove potential bias.

Paraffin-treated sections were deparaffinized and stained for 1 hr in 0.1% picrosirius red (Direct Red 80, 365548, Sigma) in picric acid solution (P6744, Sigma). Counterstaining was performed with Weigert hemotoxylin (acid condition) (HT1079, Sigma). Collagen fiber deposits and fibrillar collagen were visualized, respectively, by normal light and polarized light microscopy, and quantified with ImageJ software. Fibrilar collagen level, a direct readout of LOX family activity [8 (link), 18 (link)–20 (link)], was defined by the ratio fibrillar/quantity and calculated for statistical analysis: a low score indicates a poor level of collagen fiber organization whereas a high score indicates a high collagen fiber organization. The same methodology was used to analyze collagen fiber organization in orthotopic xenograft models and in TMAs from human PDAC.

After 70 days of culture in compression bioreactors, scaffolds were rinsed twice with PBS and fixed with 4% PFA in 10 mM CaCl₂ and 0.15 M NaCl solution for 2 h at room temperature. Samples were rinsed twice with 10 mM CaCl₂ and 0.15 M NaCl solution and cryoprotected for 2 h with 10% sucrose in 10 mM CaCl₂. Scaffolds were further cryoprotected in 30% sucrose in 10 mM CaCl₂ overnight. Scaffolds were embedded in optimal cutting temperature compound (OCT, VWR) and flash-frozen in a methanol bath on dry ice. Samples were sectioned (10–30 µm thickness) using Kawamoto’s cryofilm type 2C (SECTION-LAB Co. Ltd., Japan) using a cryotome (CryoStar NX70, Thermo Scientific) (Dyment et al., 2016 (link)). Prior to staining, sections were adhered to microscope slides (SuperFrost™ Microscope Slides, ThermoScientific) using 1% (w/v) chitosan in 1% (v/v) acetic acid. Haematoxylin (Mayer’s, Sigma-Aldrich) and eosin (Y disodium salt, Sigma-Aldrich) (H&E) staining was performed to visualize cell nuclei, cytoplasm, and extracellular matrix. Alizarin Red S staining (2 mg/mL in acetone pH 4.3) (A5533, Sigma-Aldrich) was used to stain the mineralized extracellular matrix. Picrosirius red staining (365548, P6744, Sigma-Aldrich) enabled visualization of collagen. Histological sections were imaged with an automated slide scanner (Panoramic 250 Flash II, 3Dhistech, Hungary) at ×20 magnification.