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


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.

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This study was funded by JSPS KAKENHI (Grant Number: JP18K0703708).

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Conception of the work: TN, OI, KN, YK, TM, TY. Design of the work: TN, OI, KN, YK, TM. Acquisition of data for the work: TN, KS, ET, MH, TN, TI. Analysis of data for the work: TN, KS, ET, MH, TN. Interpretation of data for the work: TN, KS, ET, TN, OI, YK, TM, TY. All authors have revised the manuscript, approved the final manuscript, and agreed to account for all aspects of the work.

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Correspondence to Teppei Nakamura.

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Animal experiments were performed in accordance with the guidelines of the Chitose Laboratory, Japan Food Research Laboratories (approval no. HK200703-01) and the Hokkaido Institute of Public Health (approval no. K30-01).

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The authors declare no competing interests.

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Nakamura, T., Sumi, K., Tsuji, E. et al. Novel polychrome staining distinguishing osteochondral tissue and bone cells in decalcified paraffin sections. Cell Tissue Res 385, 727–737 (2021).

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  • Osteoid
  • Mineralized bone
  • Cartilage
  • Bone cells
  • Osteochondral staining