Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study

A revised procedure is described for counterstaining specimens with Alcian blue for cartilage and alizarin red S for bone, and for clearing tissue with enzymes and/or potassium hydroxide. Procedures are described and suggestions are made to minimize loss or damage to bone and cartilage structures during fixation, storage in collections, and clearing and staining specimens. This method differs from previously described procedures primarily in the following: specimens are not placed in water to remove formaldehyde following fixation. Instead they are dehydrated through a series of alcohols to minimize the acetic acid activity on bony structures while staining for cartilage. Following cartilage staining, specimens are immediately deacidified in alkaline solutions instead of transferring them through several series of alcohols. Suggested variations in the method are given for staining for bone alone, for cartilage alone or for both in the same specimen.