A comparative study of the human, mouse, and zebrafish eye after staining with RGB trichrome

Eyesight plays essential roles for the survival of most animal species, and diseases leading to blindness or partial vision loss in humans have an enormous impact in life quality. The complex structure of the eye makes it ideal for the study of structure-function relationships, and to accomplish integration of histology with other disciplines, connecting basic anatomical sciences to clinical medicine. The RGB trichrome consists of the sequential staining with three dyes, fast green (as a general protein stain), picrosirius red and alcian blue (for two main components of the extracellular matrix, collagen and glycosaminoglycans respectively). Notably, this combination of primary colors matches the physiological color detection capacity of the human trichromatic eye. Application of RGB stain to human eye samples and to the eye of two widely used animal models, the mouse and the zebrafish (Danio rerio), gives rise to brilliant staining of eye connective tissues, with a range of colors from red to magenta depending on the extracellular matrix composition, and producing highly contrasted tissue interfaces, facilitating the observation of tissue structures, as well as histomorphometric analysis. Interestingly, staining of the retina resulted in differential staining of the retinal layers in the three species analyzed. These results support the effectiveness of RGB as a reliable general staining method, complementary to routine basic hematoxylin and eosin (H&E), applicable to the study of the eye in human pathology, basic preclinical research, as well as in histo(patho)logy teaching.