Picrosirius red staining and polarized light microscopy

OCT‐embedded frozen sections (10‐μm‐thick) were stained with a Picrosirius Red Stain Kit (Polysciences) according to the manufacturer's instructions. Briefly, sections were fixed in 4% formaldehyde for 10 min, stained in picrosirius red solution for 90 min, rinsed in 0.1 N HCl (2 × 1 min), and dehydrated in 70% ethanol for 30 s. The slides were then dried and mounted in Permount (Fisher Chemical). The sections were imaged under polarized light or conventional light with an Olympus BX60 microscope and the cellSens Standard 1.8.1 software (Olympus). The microscope settings (light intensity, condenser opening, exposure time, and gain) were kept constant throughout the imaging of all samples. Five images per tumor were taken and quantified with the CellProfiler software, using constant thresholding conditions enabling exclusion of background residual light and detection and quantitation of fibrillar collagen. The average picrosirius red staining intensity for each tumor is reported. The diameters (width) of fibers longer than 150 μm were measured with a similar approach as for SEM images, using the Autocontex machine‐learning method implemented in the open‐source ilastik software package to segment images (Sommer et al, 2011; Haubold et al, 2016) and the FibrilJ plugin in ImageJ (Sokolov et al, 2018).