The effects of three different demineralization agents on osteopontin localization in adult rat bone using immunohistochemistry.
Immunohistochemical localization of osteopontin, a phosphorylated acidic glycoprotein, was compared in adult rat femur fixed in 4% paraformaldehyde at 4 degrees C for 48 h and demineralized at 4 degrees C in ethylenediaminetetraacetic acid (EDTA), modified Jenkin's solution, or 15% formic acid, until radiographs indicated demineralization was complete. Formic acid was also evaluated at room temperature. EDTA solution (15 days) resulted in intense staining of osteocytes, periosteal osteoclasts and osteoblastic cells in osteonal bone. Osteoblasts were negative in the periosteum. No megakaryocyte staining was present; however, occasional neutrophils in the bone marrow were non-specifically stained. Demineralization in modified Jenkin's solution (16 days) showed osteopontin localization in bone matrix, hypertrophic and articular chondrocytes, and osteocytes. In cortical bone, almost all cement lines demarcating osteons showed very dense labeling. In the bone marrow, occasional megakaryocytes were immunopositive and neutrophils were non-specifically stained. Jenkin's produced non-specific staining of skeletal muscle and connective tissue. Formic acid demineralization (14 days, 4 degrees C) resulted in osteopontin expression in osteoblasts, osteocytes, 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 to Jenkin's demineralization. Also more intense non-specific staining of the bone marrow neutrophils was obtained than with Jenkin's. Harsh, rapid demineralization with formic acid (4 days, room temperature) produced a loss in antigenicity demonstrated by a reduction in staining intensity not experienced with the 4 degrees C protocol; however, osteopontin was still localized in bone matrix and hypertrophic zone chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Safety Assessment, Merck Research Laboratories, West Point, PA 19486.
J D
JD
Frank
R
R
Balena
P
P
Masarachia
J G
JG
Seedor
M E
ME
Cartwright
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D000941:Antigens / Q000032:analysis / Q000276:immunology; D001857:Bone Matrix / Q000378:metabolism; D001862:Bone Resorption / Q000378:metabolism; D001842:Bone and Bones / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D002113:Calcification, Physiologic / Q000187:drug effects*; D004492:Edetic Acid / Q000494:pharmacology; D005269:Femur; D005561:Formates / Q000494:pharmacology; D006851:Hydrochloric Acid; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008297:Male; D053495:Osteopontin; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000032:analysis* / Q000276:immunology; D012996:Solutions / Q000494:pharmacology; D016707:Tissue Fixation
D000085:Acetates; D000941:Antigens; D005561:Formates; D012795:Sialoglycoproteins; D012996:Solutions; C506897:Spp1 protein, rat; C030544:formic acid; D053495:Osteopontin; D004492:Edetic Acid; D019342:Acetic Acid; D006851:Hydrochloric Acid