New staining technique reliably detects collagen in fossils

Researchers have developed a new application of histological staining for screening ancient proteins, along with the microstructure of a fossil—a breakthrough in the field of paleoproteomics by addressing long-standing challenges of contamination.

A research team led by Okayama University of Science (OUS) has successfully adapted a widely used histological technique to observe the presence and distribution of ancient proteins directly within fossil tissues, preserving important microstructural context. This method provides an important tool for confirming the authenticity of endogenous (originating from ancient organisms) proteins before complex analyses, significantly increasing the reliability of paleoproteomic studies.

The results are published in Journal of Proteome Research.

The field of paleoproteomics exploits the fact that proteins are more stable than DNA and can survive in fossils for millions of years. Analyzing these ancient proteins can reveal the evolutionary relationships and biology of extinct organisms. However, traditional methods usually require crushing fossil specimens into a powder to extract the proteins. This process eliminates valuable structural information – making it impossible to determine the exact location of the protein within the tissue – and increases the risk of contamination from modern sources or microbes.

“A constant challenge in paleoproteomics is the contamination problem. It’s the question of whether or not the detected proteins are really ancient,” explained lead author Hayato Inaba, a study, education and outreach specialist at Temba City’s Dinosaur Division.

Histological Staining Detection of Collagen in Undissected Fossils for Histological Staining

To overcome these limitations, the OUS team focused on applying histological staining techniques directly to thin sections of nondemineralized fossils. Unlike methods that require demineralization (dissolving minerals with acid), which can alter tissue morphology and increase the risk of contamination, this approach preserves in situ structural information.

The researchers tested several staining methods on Pleistocene-age elephant fossils (tens of thousands of years old) recovered from the Sato Inland Sea in Japan. They identified von Giesen’s staining—a common method in modern biology—as the most effective technique for visualizing the primary protein in bone, type I collagen.

This procedure clearly turned the stained collagen bright red, allowing the team to directly map its distribution within the fossil matrix. Professor Haditsugu Sugegawa, a regenerative medicine specialist at OUS and senior author of the study, adapted this staining method for application to fossils.

Mass spectrometry supports the staining results and highlights tissue-specific protection

To confirm that the stained material was indeed ancient collagen, the team used state-of-the-art mass spectrometry in the areas that tested positive. The results confirmed the presence of type I collagen sequences identical to those of extinct elephants (proboscia), providing strong evidence that the proteins were endogenous.

The study also revealed significant differences in protein preservation between different types of fossil tissues from the same animal. While bone showed excellent collagen preservation, dentin (material-forming substance/ivory) showed almost none.

The researchers attribute this difference to the microstructure of the tissues. Dentin contains countless microscopic channels called dentin tubules, which expose the matrix more directly to the environment, accelerating collagen degradation and leaching over time. Bone, being denser, tends to preserve protein better.

“This finding provides an important guideline for future paleopartotomy research,” said Dr. Kentaro Chiba, a lecturer in the Department of Dinosaur Paleontology at OUS and the study’s corresponding author. “Understanding which tissues are most likely to retain proteins helps us select the best samples for analysis.”

A powerful screening tool for analyzing ancient proteins, including dinosaurs

This new Van Giesen staining application offers a simple yet powerful screening tool. By visualizing collagen preservation in situ, researchers can prioritize costly and complex mass spectrometry analysis of samples and have greater confidence in the validity of the results.

The research group is now looking to apply the method to older fossils, including dinosaurs, dating back tens of millions of years.

“Extracting proteins from dinosaur fossils is much more difficult because of the minute amount of remains,” noted Professor Sogegeva. “However, if we can analyze the amino acid sequences of their proteins, we can move beyond classifying dinosaurs purely by morphology. We hope to analyze dinosaurs as living organisms and get closer to understanding their physiological functions.”

Annaba concluded, “I will never forget the excitement of seeing collagen stain a vivid red color before my eyes from thousands of years ago. I hope this technology will help open the door to future discoveries, such as proteins from dinosaurs that no one has ever seen.”