不同固定和包埋方法对大鼠骨组织免疫组化结果的影响

Effects of different fixation time and embedding methods on immunohisto chemical markers of rat bone

目的探讨不同固定和包埋方法对大鼠骨组织完整性及免疫组化结果的影响。方法大鼠4只2%戊巴比妥钠麻醉,腹主动脉放血处死后立即取双下肢,室温下置于4%多聚甲醛中固定。所有标本固定后经10%乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)脱钙处理,待组织软化后进行石蜡包埋,根据固定时间和包埋方法将骨组织分为:左侧股骨整段包埋组,左侧胫骨整段包埋组,右侧股骨截断包埋组,右侧胫骨截断包埋组,各组骨组织均为4例,分别固定1、2、3和4 d,切片后行HE染色和免疫组织化学检测骨保护素(osteoprotegerin,OPG)蛋白的表达,比较各组间染色结果和OPG蛋白表达差异。结果 HE染色结果显示,整段包埋和截断包埋的骨组织骨髓腔及骨小梁周边组织结构保存完整;免疫组化结果显示,显微镜下OPG染色为深褐色细颗粒状,主要位于成骨细胞和骨髓基质干细胞胞质及细胞间质中。免疫组化染色吸光度值结果显示,整段包埋的股骨和胫骨中的OPG吸光度值,无论在固定1,2,3和4 d(4.52±0.3,4.42±0.4,8.66±0.3,0.21±0.1;6.45±0.5,6.09±0.2,9.66±0.4,0.08±0.1)均显著高于相应截断包埋的吸光度值,差异有统计学意义(P<0.05);整段包埋的股骨和胫骨吸光度值均在固定3 d(8.66±0.3,9.66±0.4)时达到最高峰值,显著高于1、2和4 d的吸光度值,差异有统计学意义(P<0.05)。结论大鼠股骨和胫骨整段包埋和截断包埋均能较好地保存骨小梁组织结构,固定3 d为相对最佳固定时间。相比股骨,胫骨组织结构能够得到更好地保存,抗原性仍较好。

Objective To investigate the effects of different fixation time and embedding methods on the immunohistochemical markers of rat bone tissue. Methods Four rats were anesthetized with 2% pentobarbital sodium and then sacrificed by abdominal aorta bleeding. The femora and tibias were moved quickly and then placed in 4% paraformaldehyde at room temperature for fixation. All bones were then treated by 10% ethylene diamine tetraacetic acid（ EDTA） for decalcification; after the tissues were softened,the bones were divided into 4 groups according to the fixation time and embedding method,left femora and tibias whole embedded in paraffin,right femora and tibias partly embedded in paraffin,there were 4 cases in each group,fixed in paraformaldehyde for 1 day,2 days,3 days,4 days,respectively. The hematoxylin-eosin（ HE） staining and expression of osteoprotegerin（ OPG） protein was detected by immunohistochemistry in paraffin section. Results HE staining showed that the histological structure of whole embedded bone marrow cavity and trabecular bone was well preserved. Immunohistochemistry showed that the OPG positive staining was brown and mainly located in the osteoblasts,bone marrow stromal cells and cytoplasm. OPG absorbance value of the whole embedded femur and tibia（ 4. 52±0. 3,4. 42±0. 4,8. 66±0. 3,0. 21±0. 1; 6. 45±0. 5,6. 09±0. 2,9. 66±0. 4,0. 08±0. 1） was significantly higher than that of the partly embedded at 1 day,2 days,3 days and 4 days after fixation（ P〈0. 05）. The absorbance of whole embedded femur and tibia reached the peak value at 3 days（ 8. 66±0. 3,9. 66± 0. 4 after fixation,which was significantly higher than that at 1 day,2 days and 4 days（ P〈0. 05）. Conclusions Trabecular tissue structure can be well preserved in whole embedded and partly embedded femur and tibia. Three days for fixation were the best time. Tissue antigenicity and structure of tibia were better preserved compared to femur.