Visualizing the Osteocyte Lacuno-Canalicular System via a Rapid 10-Minute Silver Nitrate Staining Method

The conventional Ploton silver method employs a high-concentration 50% (w/v; 2.943 mol/L) silver nitrate solution for histological staining and characterization of the osteocyte lacuno-canalicular system (LCS). However, it is limited by prolonged staining times (55 min) and by risks of LCS ultrastructural damage and/or incomplete impregnation. To address these limitations, we developed the Wu-Wang silver nitrate staining method, which uses a 1 mol/L silver nitrate solution under elevated temperature (50-70 °C) to achieve rapid, effective, and high-contrast visualization of the osteocyte LCS within 10 min. We further demonstrate that this novel method enables robust LCS visualization across multiple vertebrate species. Compared with the Ploton method, the Wu-Wang method substantially reduces staining time and overcomes staining limitations inherent to prolonged exposure to concentrated silver nitrate solutions. This rapid and efficient staining method supports more accurate quantitative analysis of LCS morphology and facilitates systematic investigation of osteocyte and LCS morphogenesis, as well as the pathological mechanisms underlying bone and joint disease. Key features • A novel rapid method uses 1 mol/L silver nitrate at 50-70 °C, enabling high-contrast LCS staining within 10 min. • A rapid silver nitrate staining method provides superior, consistent LCS visualization across multiple vertebrate species. • The novel method serves as a fast, efficient histological tool to investigate osteocytes, LCS, and bone-joint pathology.