Novel polychrome staining distinguishing osteochondral tissue and bone cells in decalcified paraffin sections

Abstract

The bone is a dynamic and metabolically active organ in which growth and resorption of the osteochondral matrix is orchestrated by osteoblasts and osteoclasts. For decalcified paraffin-embedded specimens, decalcifying agents alter the staining intensity, and excess decalcification interferes with bone staining.Robust bone staining methods independent of the decalcification conditions and animal species are lacking.In this study, we have developed a novel polychrome staining method, named JFRL staining, which stains the components of osteochondral tissue in different colors. With this staining we could visualize the hyaline cartilage as blue by alcian blue, osteoid as red by picrosirius red, and mineralized bone as green by picro-light green SF or picro-naphthol green B and easily distinguished osteoblasts, osteocytes, and osteoclasts. In mineralized bone, this staining revealed the obvious lamellar structures and woven bone.Notably, this staining was independent of the decalcification conditions and experimental animal species examined. To verify the usefulness of JFRL staining, we observed cotton rat tail which has shorter length and shows a false autotomy. The caudal vertebrae were normally developed via endochondral ossification without a fracture plane. At 6 months of age, the number of chondrocytes declined and the hypertrophic zone was absent at the epiphyseal plate, which might reflect the shorter tail. In conclusion, JFRL staining is the first method to simultaneously distinguish osteochondral matrix and bone cells in one section regardless of decalcifying conditions. This robust staining will provide new information for a wide number of biomedical fields, including bone development, physiology, and pathology.

Rat bone marrow mesenchymal stem cells induced by rrPDGF-BB promotes bone regeneration during distraction osteogenesis

“…The decalcified samples were cleaned and then gradually dehydrated with alcohol, after which they were immersed in xylene (Sigma, Switzerland) and finally embedded in paraffin (Leica, Germany) ( Guo et al, 2020 ; Nakamura et al, 2021 ; Marinopoulos et al, 2022 ). The samples were frozen at −20°C for 8 h, and then cut into 3.0 μm thick sections with a microtome (RM2135, Leica, Germany), removed with adhesion microscope slides (Citotest, Jiangsu, China) and baked in a 60°C oven.…”

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

“…There are other protocols for decalcified samples, for example, Goldner’s trichrome, toluidine blue and picrosirius red staining. However, these methods have limitations in staining outcomes for tissue differentiation and quantification, and some also involve multiple chromogens and complicated processing steps [ 6 , 9 , 14 ].…”