By: Brooke H. Dubansky, PhD, HTL(ASCP)CMHCM
Reptiles have always played major roles in human culture, economics, and scientific endeavors. Reptile skin, in particular, has fascinated the scientific community for centuries. The large variety of different scale patterns, shapes, and colors have been used by herpetologists to delineate species, by anatomists to interpret behavior and mechanics in living and fossilized animals, and even evolutionary biologists have used reptile scales to reconstruct the evolutionary history of bird feathers and the origin of flight (see Figure 1 for various scales in different reptile species). While there are many reasons to perform histotechniques on reptile skin, our labs in particular are interested in using histology and other anatomical techniques to 1.) Provide a functional interpretation of skin microstructure to understand reptile feeding behaviors, and 2.) Use normal developmental processes in reptile skin as models for studying some human skin pathologies and other disorders.
Applying histotechniques to reptile integument poses special challenges that mostly affect microtomy. These challenges arise from the fact that scales are a diverse mixture of hard and soft tissues.
The outer layer of the skin, called the epidermis, is unique in reptiles compared to mammals, in that it contains a special type of hard keratin that makes it impermeable to processing fluids and paraffin; this often causes the surface of the scale to break away from the paraffin block during sectioning. Once the epidermis breaks away from the block, it resists adherence to the slide and often folds over and obscures other parts of the section (Figure 2). This property of reptile scales makes them similar to (but not quite the same as) fingernails and toenails often encountered in dermatopathology laboratories. We cannot change the keratin properties in the reptile epidermis, and increasing processing or infiltration times will not correct the problems described. Instead, our lab has developed special coverslipping techniques that correct and flatten the folds retroactive to the development of these artifacts. In reptiles that shed their skin, additional challenges may arise if the epidermis becomes detached from the rest of the skin during certain phases of the shedding cycle.
The dermis lies underneath the epidermis, and is composed of layers of dense connective tissue. Compared to humans and other mammals, the collagen fibers that comprise the dermis in reptiles are often highly organized with orthogonally arranged layers, creating an incredibly dense, compact tissue. In several species of crocodilians and lizards, the dermis may develop bony deposits called osteoderms. It is not always possible to detect bone in the scales before processing the specimen for paraffin embedding and microtomy, especially if the animal is young and/or small. If discovered while sectioning, small osteoderms can be dealt with by using a surface decalcification procedure. This technique involves soaking the cut surface of the block in 5-10% formic acid for around 10-15 minutes. After rinsing the block and returning it to the microtome, a few sections can be obtained before needing to repeat the procedure.
Once challenges with sectioning and mounting the skin samples are overcome, staining procedures traditionally used for mammalian tissues can be used without modification in most cases. Some of the most common stains we use in our labs for reptile skin include Masson Trichrome, Weigert Resorcin Fuchsin, and Picrosirius red to assess collagen and/or elastin; and the Yoshiki-Nikaido-Yamazaki-Tachikawa (YNYT) Silver stain for osteoid and unmineralized bone matrix to analyze osteoderm structure and development (Figure 3). The Masson Trichrome procedure will be familiar to most practicing Histotechs. Weigert Resorcin-Fuchsin is very similar to the Verhoeff Elastic stain, only Resorcin-Fuchsin is used to stain the elastic fibers instead of a solution of alcoholic hematoxylin, ferric chloride, and iodine.
Picrosirius red combines the dye Sirius Red with saturated picric acid. This combination enhances the birefringent properties of collagen, and different collagen types (I-III) will emit different colors from red to yellow to green when viewed with polarized light. This stain will also differentiate unmineralized bone matrix (osteoid) from immature mineralized bone matrix.
The YNYT stain is a silver nitrate stain used to enhance the contrast between calcified bone matrix and unmineralized bone matrix. The tissue is treated with a silver nitrate solution en bloc, after fixation and before decalcification and processing. The sliver nitrate precipitates in the calcified matrix and the addition of cyanuric chloride enhances the eosinophilia of osteoid. The result is bright pink osteoid that contrasts sharply with adjacent calcified bony matrix (stained with variations of black, brown, and red).
These classic stains are beautiful and ideal for creating maximum contrast between dermal collagen and other structures that may be present in the reptile dermis such as bone, cartilage, blood vessels, pigments, and sensory organs. To learn more about the histotechniques we use for reptile skin in our labs, see our recently published article in the current issue of The Journal of Histotechnology.