Fin regeneration and fracture assay to visualize and quantify live bone formation and repair. (A) Schematic representation of a zebrafish with a standard fluorescent stereomicroscope image of a live Alizarin red S (AR) pre-amputation caudal fin (inset). (B) Schematic representation of bone regeneration after fin amputation showing the (simplified) cascade of events that follow after fin amputation to regenerate bone (a single ray depicted here). This allows studying de novo bone formation by newly formed osteoblasts (orange cells) and differentiated osteoblasts (green cells) and subsequent remodeling by osteoblasts and osteoclasts (purple cells) in an adult fish. Note that during osteogenesis that there is a gradient of mineralization. (C) Live images of the tail fin labeled with Alizarin red (red) prior to amputation (i, ii) and Calcein (green) post-amputation (iii, iv) taken on a fluorescent dissecting microscope. All images in panel come from the same fish. Seven days post-amputation showing regrowth of new bone (green). Note that intense Calcein staining is visible distally from the amputation site (white dotted line). (D) The fracture healing assay involves applying pressure on a fin ray bone element to induce a small fracture to one segment of the fin ray (i), which is visible with life AR staining (ii). Green Calcein labels the new bone formed in the fracture callus by 7 days (iii and iv). The white arrow indicates the fracture site. Scale bars = 500 μm, 3 months old wildtype TL/EKK females.
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