THE EFFECTS OF 3 DIFFERENT DEMINERALIZATION AGENTS ON OSTEOPONTIN LOCALIZATION IN ADULT-RAT BONE USING IMMUNOHISTOCHEMISTRY

Immunohistochemical localization of osteopontin, a phosphorylated acid ic glycoprotein, was compared in adult rat femur fixed in 4% paraforma ldehyde at 4-degrees-C for 48 h and demineralized at 4-degrees-C in et hylenediaminetetraacetic acid (EDTA), modified Jenkin's solution, or 1 5% formic acid, until radiographs indicated demineralization was compl ete. Formic acid was also evaluated at room temperature. EDTA solution (15 days) resulted in intense staining of osteocytes, periosteal oste oclasts and osteoblastic cells in osteonal bone. Osteoblasts were nega tive in the periosteum. No megakaryocyte staining was present; however , occasional neutrophils in the bone marrow were non-specifically stai ned. Demineralization in modified Jenkin's solution (16 days) showed o steopontin localization in bone matrix, hypertrophic and articular cho ndrocytes, and osteocytes. In cortical bone, almost all cement lines d emarcating osteons showed very dense labeling. In the bone marrow, occ asional megakaryocytes were immunopositive and neutrophils were non-sp ecifically stained. Jenkin's produced non-specific staining of skeleta l muscle and connective tissue. Formic acid demineralization (14 days, 4-degrees-C) resulted in osteopontin expression in osteoblasts, osteo cytes, osteoclast precursors, bone matrix, osteoid, cement lines, and chondrocytes; osteoclasts, although present in very low numbers, were also positive. More labeled osteoblasts could be identified compared t o Jenkin's demineralization. Also more intense non-specific staining o f the bone marrow neutrophils was obtained than with Jenkin's. Harsh, rapid demineralization with formic acid (4 days, room temperature) pro duced a loss in antigenicity demonstrated by a reduction in staining i ntensity not experienced with the 4-degrees-C protocol; however, osteo pontin was still localized in bone matrix and hypertrophic zone chondr ocytes. These results indicate that demineralization is compatible wit h retention of immunoreactive osteopontin in adult rat bone. Both EDTA and formic acid demineralization produce excellent immunostaining and are preferred over the modified Jenkin's solution to minimize backgro und levels of nonspecific staining.