Transmission electron microscopy analysis of collagen fibril size

Mouse ankle and knee joints were dissected and fixed in fresh 1.5% glutaraldehyde/1.5% PFA (Tousimis) with 0.05% tannic acid (Sigma) in 1 × PBS at 4°C overnight to preserve the native tension on relevant tendons. The next day, flexor digitorum longus (FDL), Achilles, and patellar tendons were dissected out in 1 × PBS, and placed back into fixative. Samples were then post-fixed in 1% osmium tetroxide (OsO₄), rinsed in Dulbecco’s Modified Eagle Medium (DMEM), and dehydrated in a graded series of ethanol to 100%. Samples were rinsed in propylene oxide, infiltrated in Spurrs epoxy, and polymerized at 70°C overnight. TEM images were acquired using an FEI G20 TEM at multiple magnifications to visualize transverse and longitudinal sections of collagen fibrils. Collagen fibril diameter was measured using the Fiji release of ImageJ (Schindelin et al., 2012).