A whole-mount method for trypsin clearing and collagen type II antibody staining

Although cartilage and bone are often stained to study development in fishes, collagen forms before either of these tissues during skeletal ontogeny. We describe a new method that combines conventional trypsin clearing of whole-mount specimens with staining of Collagen Type II using antibodies. Specimens were fixed briefly in paraformaldehyde, digested in a trypsin solution, bleached with hydrogen peroxide, permeabilized with Proteinase K, antigen labeled with primary and secondary antibodies, and stored in glycerol. This method makes both cellular and acellular collagen visible under ultraviolet light with limited background staining. Specimens showed no signs of damage from any of the solutions used for staining, but the length of time for fixation appears to be important. This method permits visualization of collagen condensation prior to cartilage formation in endochondral bone and can be used to study evolution of skeletal ontogeny and to develop new skeletal characters for phylogenetic analysis.