New Application of Histological Staining for Visualization of Endogenous Proteins in Fossil Material

Biochemical applications are increasingly utilized in paleontological studies, especially for detecting ancient proteins in fossil samples. Histopathological staining techniques have been applied, but they have yet to specifically target type I collagen, the primary bone matrix protein and the most significant protein of interest in paleoproteomic research. Moreover, these staining methods are often applied to demineralized fossils, which remove the original microstructure of the bone matrix and increase the risk of contamination. To address these limitations, this study aimed to test the effectiveness of special staining methods for detecting collagen using Pleistocene-aged fossil specimens. Trials on demineralized and nondemineralized modern bone samples, as well as nondemineralized fossil samples, demonstrated Van Gieson's staining method as the most suitable for visualizing collagen distribution in hard tissue matrices. Colorimetric analysis, electrophoresis, and subsequent mass spectrometry of extracts further confirmed the endogenous nature of the collagen in the fossil samples. Future studies may benefit from employing Van Gieson's staining on nondemineralized bone samples to detect collagen in fossils, advancing our understanding of ancient protein preservation.