Double whole‐mount staining for bone deposition and resorption

Methods to detect osteoblasts, bone depositing cells, and osteoclasts, bone resorpting cells are known. These methods involve enzymatic procedures (alkaline phosphatase and tartrate‐resistant acid phosphatase) and are usually conducted on sectioned material; with the procedure for each type of staining conducted on adjacent serial sections. We have optimised a procedure to whole‐mount stain the same zebrafish for both bone deposition and resorption. The alkaline phosphatase procedure stains osteoblasts a purple colour and the tartrate‐resistant acid phosphatase gives a red colour; making the two cell types easily distinguishable. The stained specimen can be decalcified if necessary and sectioned, with preservation of the cell staining. We have applied this methodology to whole fish at various stages of development and have focused our investigations on the jaw, fins and vertebral column regions. Bone deposition was clearly observed in the mandible and maxilla; the developing fins, the ends of fin ray segments and the edges of the developing calvariae. On similarly aged fish, bone resorption was detected in the neural spines of the vertebrae; mandible and maxilla; with very limited amounts within the fins. We believe this methodology would be useful to the field of skeletal biology as it enables double staining of the same specimen and avoids lengthy resin embedding procedures. Funding NSERC, Canada Grant Funding Source NSERC, Canada