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.

The formation of the caudal vena cava is a complex process involving development, regression, and anastomosis. In mammals, the normal caudal vena cava runs to the right side of the abdominal aorta, while duplication of the caudal vena cava has been identified as a congenital abnormality in both companion animals and humans. The present study demonstrates that Slc:Hartley guinea pigs frequently possess asymptomatic duplicated caudal vena cava. The prevalence was 30% and 24% for males and females, respectively, with no sex-related differences. In accordance with Saad et al. (2012)’s criteria, duplicated caudal vena cava were classified into two distinct variations. The dominant variation was a complete duplication without iliac anastomosis where the left caudal vena cava continued from the left common iliac vein and joined the left renal vein; the left renal vein ran to the right to join the right caudal vena cava. The alternative variation was an incomplete duplication where the left caudal vena cava joined the right infrarenal caudal vena cava at a more cranial point than in normal cases; the renal segment was unchanged. Iliac anastomosis was not found in any cases. Duplicated caudal vena cava neither affected the body weight nor the kidney weight. In conclusion, Slc:Hartley guinea pigs frequently possess asymptomatic duplicated caudal vena cava in the absence of iliac anastomosis and appear to be a novel and useful animal model for duplicated caudal vena cava in animals and humans.