A New Method for Staining Ligaments and Tendons of Small Vertebrates

Clearing and staining techniques are widely used to study the structure of small vertebrates. These techniques consist mainly of soft tissue clearance by enzymatic or caustic digestion and staining of bone, cartilage, or nerves. However, although there is an increasing need to describe other soft-anatomy structures, such as ligaments and tendons, the methods available to visualize these tissues are a challenge that requires very fine dissections and three-dimensional reconstructions using tissue sections or microtomography. In this paper we present a simple and economical method for staining fibrous connective tissues, particularly ligaments and tendons. The method begins with an enzymatic proteolysis that prevents unselective soft tissue degradation. Then the bones are stained to facilitate understanding the structure associated with the bone tissue of interest. Subsequently, the connective tissues are stained in two steps, first with a deep stain and then with an alcoholic differentiation. The final products are stored in glycerol. Our technique has the advantages of being as simple and short as traditional staining techniques of bone and cartilage, requires inexpensive materials and equipment, and is applicable to specimens preserved in museums. We demonstrate that our high-performance method works well with specimens of the major vertebrate groups. This approach will facilitate anatomical description of the musculoskeletal system, helping morphological, functional, and taxonomic analyses.