Acid-Free Staining Procedure to Demonstrate Nerves in Whole Vertebrate Specimens with the Differentiation of Bone and Cartilage

The value of clearing and staining whole organisms to study vertebrate anatomy is unquestionable. These methods have been developed for over a century leading to protocols to prepare triple-stained specimens to differentiate between bones, cartilage, and nerves. Despite their potential to advance the field of comparative anatomy, nerve-staining methods have been used by a small number of vertebrate systematists in part because of the inconsistently successful preparations. Here, we report on several modifications to the current Sudan black B protocols and propose a new acid-free protocol to differentiate among bone, cartilage, and nerves in whole small vertebrates. This method may also be used to stain solely bone and cartilage by eliminating the nerve-staining steps. The technique herein described is successful for preparing juveniles and adults (including miniatures).