The study, published as an open-access technical note in the International Journal of Legal Medicine, was carried out by Simona A. Dinu, Iris Sluis, Wilma Duijst and Tristan Krap, with bones obtained through the body donation program of Amsterdam University Medical Centers. The team worked with humeri, tibiae and femora from four donors: two women aged 80 and 84 whose bones had been stored in closed buckets at 4 degrees Celsius for one to two years, a 91-year-old man whose bones were processed within three days of death, and a 78-year-old man whose body was exhumed after two years of burial. These three categories, fresh, controlled aged and taphonomically altered, allowed the researchers to test whether their methods could survive contact with real-world decomposition.

The preparation method builds on a classic manual technique originally described by Frost in 1958 and later updated by Maat and colleagues. Bone pieces roughly two millimeters thick are cut with a handsaw, then wet-ground on waterproof sanding paper attached to a glass slab until translucent. But the team found two crucial improvements were needed. First, polishing with fine P1000 sandpaper proved insufficient to remove the scratches left by coarse grinding, so they recommend finishing with P2000 grit. Scratches matter enormously in stained specimens because dye particles tend to lodge in them, obscuring the very structures researchers want to see. Second, the team added an ultrasonication step: bone slices were submerged in tap water with a few drops of dish soap and ultrasonicated for two hours to flush microscopic grinding debris out of the Haversian canals. Without this step, debris particles bind dyes eagerly and produce specimens cluttered with artifactual coloration around the canals.

Another key innovation is a brief acid etching step adapted from a protocol by Osborne and Curtis. Before some staining procedures, slices are submerged in 1 percent formic acid for five minutes, rinsed, then placed in 70 percent ethanol for twenty minutes. This short, low-concentration treatment roughens the mineral surface and exposes the underlying protein matrix, giving dyes more contact area to bind, without decalcifying the tissue, which would require far longer exposure. The effect was dramatic: without acid etching, Hematoxylin and Eosin, Sirius Red and Fast Green, and Toluidine Blue all produced poor or nearly featureless coloration. With etching, the same dyes yielded crisp, reproducible images. Interestingly, for Masson’s Trichrome and Azocarmine G, which already stain well, etching caused overstaining and actually reduced the amount of visual information available.

The five stains were chosen from a 2025 review by Sluis and colleagues that compiled 45 dyes potentially suitable for anthropological applications. Three of them, Hematoxylin and Eosin, Toluidine Blue and Azocarmine G, were selected for their ability to reveal microstructure. H&E enhanced the concentric and interstitial lamellae that are barely visible in unstained ground sections, and selectively colored some osteons and cement lines. Toluidine Blue proved excellent for visualizing cement lines with minimal background, clearly delineating the contours of osteons and also coloring the edges of osteocyte lacunae. Azocarmine G, which binds acidic cellular components, distinguished osteons sharply from adjacent interstitial bone and stained them uniformly from the Haversian canal to the cement line. The remaining two stains, Sirius Red and Fast Green and Masson’s Trichrome, target composition: Sirius Red binds collagen while Fast Green binds non-collagenous proteins, and Masson’s Trichrome similarly separates intact collagen from denatured collagen and other proteins.

The compositional stains produced the most striking findings. Fresh bone stained predominantly red with Sirius Red and Fast Green, consistent with its high collagen content, while the controlled aged and taphonomically altered bones stained predominantly green, indicating that most collagen had already degraded during the two-year storage or burial periods. This result contrasts with earlier studies by Jellinghaus and colleagues, who found that human bones buried for up to 45 years still retained substantial collagen. The authors suggest several possible explanations: the controlled aged bones were not intact, leaving them more exposed to degradative factors, and climate conditions at the deposition site can dramatically accelerate decomposition, with early taphonomic changes exerting the strongest influence on the bone’s diagenetic trajectory. Masson’s Trichrome told a complementary story, with red and violet regions signaling non-collagenous proteins appearing within some osteons and at the outer edges of taphonomically altered slices, precisely where exposure to the burial environment is greatest.

The microstructural stains also revealed biologically meaningful patterns. Some osteons showed much stronger affinity for the dyes than others, and the researchers attribute this to their newer formation: young osteons contain more of the organic protein matrix, including acidic proteins like osteopontin and bone sialoprotein produced by osteoblasts, and these osteons tend to sit toward the periosteum where bone formation outpaces resorption. Toluidine Blue’s staining of cement lines is particularly valuable because cement lines are rich in negatively charged sulfated mucosubstances that interact strongly with this cationic dye, and the dye’s effectiveness depends critically on the pH of the solution. Tracing cement lines allows researchers to follow remodeling events through the bone, and the extent of remodeling correlates with age, mechanical stress and lifestyle, information that can support, though never replace, conventional age-at-death estimation.

The practical advantages of the approach are considerable. Because fixation, decalcification and embedding are all eliminated, fixation being optional and decalcification and embedding avoided entirely, a stained bone sample can be prepared within a single day rather than over several weeks. The mineral matrix is preserved as a background against which changes in the organic portion of the bone can be read, which is especially important for specimens in advanced decomposition where most organic material has already degraded. The method is inexpensive, uses low-hazard chemicals, and requires relatively little training. The authors also demonstrated reproducibility: specimens prepared in duplicate and stained by two separate researchers showed little to no variation, and results were consistent across staining sessions separated by more than a year, and across humeri, tibiae and femora.

The researchers are candid about the limitations of the manual grinding method. Slide thickness cannot be precisely controlled, which produces variations in brightness and makes even focusing difficult, and handling artefacts such as cracks risk being misclassified as genuine taphonomic alterations, potentially leading to wrong conclusions about how degraded a sample really is. Mechanical stress from grinding could also affect the organic matrix in samples with poor collagen quality. Precise instruments like diamond blade saws could solve some of these problems but at considerably higher cost. The team also notes that image acquisition itself needs standardization, since two different microscope cameras produced noticeably different color renderings of the same slides, and inconsistent white balance or exposure could skew interpretation of color patterns in future studies.

Overall, the study represents a first step toward making histochemistry a standardized tool in forensic and anthropological casework. By demonstrating that five widely available stains can be reliably applied to unfixed, undecalcified, unembedded human bone, whether fresh, refrigerated or altered by two years of burial, the researchers have laid the groundwork for adapting many more of the 45 candidate dyes identified in their earlier review. Combined with automated image analysis, which could reduce the subjectivity that currently ties structure identification to individual researcher experience, stained bone histology could eventually support age and lifestyle predictions and post-mortem interval estimation for skeletonized remains, turning the chemistry of dyes into a new language for reading the dead.

Subject of Research: Histological staining protocols for unfixed, undecalcified human bone in forensic anthropology

Article Title: A technical note on the preparation and staining of unfixed, undecalcified and unembedded fresh and taphonomically altered human bone with five common stains and dyes

Article References: Dinu, S. A., Sluis, I., Duijst, W., & Krap, T. (2026). A technical note on the preparation and staining of unfixed, undecalcified and unembedded fresh and taphonomically altered human bone with five common stains and dyes. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03986-9

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03986-9

Keywords: forensic anthropology, bone histology, histological staining, human bone, taphonomy, collagen degradation, Hematoxylin and Eosin, Sirius Red, Toluidine Blue, Masson's Trichrome, Azocarmine G, post-mortem interval