Tumor Histology and Collagen Imaging

10 to 12 week old tumors were dissected and fixed in 10% neutral buffered formalin overnight at room temperature. They were then embedded in paraffin and sectioned in 5–10 um sections. In some cases, sections were stained with H and E, picosirius red, or trichrome stain prior to SHG imaging. Prior staining had no effect on SHG signal. Images were acquired on a Zeiss LSM 880 Airyscan confocal microscope using an inverted, motorized Zeiss Axio Observer Z1 frame. Two-photon images were collected at 880 nm, using non-descanned detectors set to 440 nm for SHG. Three to four z-stacks were acquired (step size 2 um) per tumor. The z-stacks were compressed and TACS signature was scored by three blinded reviewers as previously described (Corsa et al., 2016 (link)) (Provenzano et al., 2006 (link)).