Recovery and Enhancement of Faded Cleared and Double Stained Specimens

Cleared and stained specimens may become faded and/or opaque after long periods in preservative solutions. We developed a technique to revitalize faded and/or destained specimens regardless of their age. By adapting and revising Wassersug's procedure for differential staining of bone and cartilage, we successfully cleared and restained numerous small amphibian specimens of various ages. After rehydrating, specimens were bleached in potassium hydroxide and hydrogen peroxide, stained with alcian blue, and macerated with trypsin as needed. Alizarin red stain was applied or reapplied, although staining occasionally was unsuccessful in old formalin fixed specimens. After restaining, the specimens were dehydrated and placed in glycerol.