Juvenile cornsnake (Pantherophis guttatus) that was cleared and stained last year. The Alcian blue excess was not completely removed by the KOH and we probably didn't use enough alizarin.
Basically, most protocols are derived from Dingerkus and Uhler (1977), although this technique is much older than that, and much details can be found in "Clearing and staining skeletons of small vertebrates", by Davis, Gore & Osgood, and which is available here.
Basically, tissues undergo an enzymatic digestion using trypsin. This is the clearing stage. Then the specimen is double stained, first using Alcian blue, which stains cartilage blue, then with Alizarin red, which stains bone a deep red colour. Specimens are then stored in a glycerin solution, which has an index of refraction very close to that of collagen, which helps "transparize" the specimen further.
In addition to looking truly beautiful, this is a very useful technique in biology, still widely in use today. It allows the study of the skeleton of very small vertebrates without the need for "classical" osteological preparations, which are especially problematic with this kind of material. It also routinely used for the study of ontogenetic patterns of ossification, along with more modern techniques such as X-ray microtomography. A third staining agent, Sudan black B is also sometimes used as a nerve stain on previously cleared and double-stained specimens.
While on the subject, check out these specimens, cleared and stained by Adam Summers:
Some really cool fish
A friend of mine recently sent me a sample of a product called visikol, which is basically a clearing agent used for microscopy. I've yet to give it a try (which I should do very soon), but I've seen some very interesting preliminary results. It seems to clear the specimens very quickly, the tricky part being the staining process. Will probably post the results over here when I start experimenting with it.
Juvenile black scorpionfish (Scorpaena porcus) recently finished. This one took didn't clear well, probably due to the improper use of trypsin. However the stains worked quite well. Notice how the scales are deeply stained by the Alizarin red.
Close up on the jaw of a juvenile skate (Raja sp.). The tesserae are clearly visible
Reference:
Dingerkus G, Uhler L. 1977. Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technology, 52: 229-232.
