Clearing and Staining Fishes (equal parts science & art)

If I told you that every fish that I have cleared and stained looked like the Echiostoma tanneri (AMNH 215419) specimen pictured in this post, I would be lying. In truth, the process of first clearing the flesh of vertebrates with a trypsin solution and then staining their bones with alizarin red and cartilage with alcian blue, can have variable effects depending on a great number of factors. Examples include the initial state of preservation, specimen size and fragility, age and reactivity of trypsin used, concentration of dyes, and many others.

Captured 2b

Whatever the case, the best of course of action when first learning to clear and stain fishes is to take a conservative approach at all stages. While I was working at the AMNH, I started on a project clearing and staining large number of specimens in the deep sea order of fishes, Stomiiformes. This group of fishes, of which E. tanneri is member, were a particular challenge; their bodies are extremely fragile and their bones weakly ossified. It took great care to keep many of them from completely disintegrating during the clearing process and for stain to be sufficiently visible on some of the thinest bone you will ever see in adult fishes. Probably the most helpful advice that I was ever given while working on this project, and one that is relevant to clearing and staining in general, was from Radford Arrindell (collection staff at AMNH Ichthyology Department). He reminded me that in clearing and staining, you can always go back a step (or steps). What this means is that if you don’t leave a specimen in tripsin long enough, you can always go back after you stain the bone to let the specimen clear a bit more. However, if you leave that specimen in too long and it starts to fall apart, you can never put it back together. I can go on forever on advice for each stage of clearing and staining, and I may do that some day, but for now I will leave you with the advice to get a trusted clearing and staining protocol (my personal favorite is Pothoff 1984) and take it slow!

Reference: Pothoff, T. 1984. Clearing and staining techniques. In H.G. Moser (editor), Ontogeny and systematics of fishes. Special Publication of the American Society of Ichthyologists and Herpetologists. No. 1: 35–37. Lawrence, KS: Allen Press.