Frank J D, Balena R, Masarachia P, Seedor J G, Cartwright M E
Department of Safety Assessment, Merck Research Laboratories, West Point, PA 19486.
Histochemistry. 1993 Apr;99(4):295-301. doi: 10.1007/BF00269102.
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)
对骨桥蛋白（一种磷酸化酸性糖蛋白）进行免疫组织化学定位，比较其在4℃下用4%多聚甲醛固定48小时的成年大鼠股骨中的情况，然后在4℃下用乙二胺四乙酸（EDTA）、改良的詹金氏溶液或15%甲酸进行脱钙，直至X线片显示脱钙完成。甲酸也在室温下进行了评估。EDTA溶液（15天）导致骨单位骨中的骨细胞、骨膜破骨细胞和成骨细胞强烈染色。骨膜中的成骨细胞呈阴性。未出现巨核细胞染色；然而，骨髓中偶尔的中性粒细胞有非特异性染色。在改良的詹金氏溶液中脱钙（16天）显示骨桥蛋白定位于骨基质、肥大和关节软骨细胞以及骨细胞中。在皮质骨中，几乎所有界定骨单位的黏合线都显示出非常密集的标记。在骨髓中，偶尔的巨核细胞呈免疫阳性，中性粒细胞有非特异性染色。詹金氏溶液导致骨骼肌和结缔组织出现非特异性染色。甲酸脱钙（4℃，14天）导致骨桥蛋白在成骨细胞、骨细胞、破骨细胞前体、骨基质、类骨质、黏合线和软骨细胞中表达；破骨细胞虽然数量很少，但也呈阳性。与詹金氏溶液脱钙相比，可以识别出更多标记的成骨细胞。与詹金氏溶液相比，骨髓中性粒细胞的非特异性染色也更强。用甲酸进行剧烈、快速脱钙（室温，4天）导致抗原性丧失，表现为染色强度降低，这是4℃方案未出现的情况；然而，骨桥蛋白仍定位于骨基质和肥大带软骨细胞中。（摘要截断于250字）
