Multicolor Histochemical Staining for Identification of Mineralized and Non-Mineralized Musculoskeletal Tissue: Immunohistochemical and Radiological Validation in Decalcified Bone Samples

Histochemical staining of paraffin-embedded decalcified bone samples is commonly used in preclinical research of musculoskeletal diseases, enabling the visualization of multiple tissue components by the application of chromogens. The purpose of this study was to introduce a novel multicolor staining protocol involving optimized chemical reagents and procedure, allowing the identification of high-mineralized bone, low-mineralized fracture callus, cartilage and skeletal muscle fibers simultaneously. Fractured femur and healthy tail vertebra samples from adult male Sprague–Dawley rats were decalcified with EDTA and formic acid, respectively, followed by paraffin embedding, tissue sectioning and multicolor staining. Conventional Movat’s pentachrome and safranin O / fast green staining were conducted in parallel for comparison. Immunohistochemical staining of collagen type-X and micro-CT analysis were included to further validate the efficacy of the staining method. The multicolor staining allowed visualization of major musculoskeletal tissue components in both types of decalcified samples, providing quality outcomes with fewer chemical reagents and simplified procedures. Immunohistochemical staining demonstrated its capacity for identification of the endochondral ossification process during fracture healing. Micro-CT imaging validated the staining outcome for high-mineralized skeletal tissue. The application of the multicolor staining may facilitate future preclinical research involving decalcified paraffin-embedded samples