An Optimized Staining Method for Visualization of Thermally Denatured Dermal Collagen

Accurate visualization of thermal damage zone (TDZ) in the dermis is critical for energy-based device (EBD) and burn research. While nitroblue tetrazolium chloride (NBTC) staining and birefringence imaging are commonly used, both rely on detecting loss-of-signal, thereby only inferring TDZ indirectly. Other methods, such as hematoxylin and eosin and Masson's trichrome, are protocol-sensitive and limited in sufficiency and consistency for TDZ visualization. This study aims to validate a novel staining protocol optimized for direct and selective visualization of thermally denatured collagen, termed Ahn-van Gieson stain (AVG).

Protocol analysis was performed to identify sources of inconsistency in the iron hematoxylin-based Verhoeff-van Gieson stain, followed by comparison of multiple versions of modifications for optimization. Horizontal sections of the human dermis were irradiated with a defocused CO₂ laser beam under thermal recording with a 10.6 μm notch filter. Thermal data were processed to generate peak temperature and Arrhenius integral maps for collagen denaturation. Slides stained with the optimized AVG protocol were overlaid with thermal maps for validation.

Iron-hematoxylin stained both TDZ and surrounding collagen regions, but subsequent differentiation using FeCl₃ led to partial loss of staining within TDZ, contributing to inconsistent results. Replacing the FeCl₃ differentiation step with 1 min 1% acid alcohol treatment produced the most consistent and high-contrast visualization of TDZ. The protocol required approximately 40 min, functioned reliably on both frozen and paraffin-embedded samples, and was compatible with birefringence imaging. The stained areas corresponded well with thermal maps for collagen denaturation, supporting the specificity of AVG for thermally denatured collagen.

The novel protocol enables direct, high-contrast, and reproducible visualization of thermally denatured collagen using standard equipment in a short time. It holds promise as a practical histological tool in EBD and burn research, where precise collagen damage visualization is essential.