冰冻切片免疫荧光中组织固定方法研究

目的通过对3种不同固定液对肝组织冰冻切片免疫荧光过程中固定情况的比较,探寻保存抗原最佳固定效果的方法。方法取Wistar大鼠肝脏,OCT包埋后置于冰冻机切片,片厚8～10μm。分别用10%甲醛、丙酮和1%多聚甲醛3种固定液固定后,进行免疫荧光染色,经Image-pro图像分析软件及单因素方差分析统计,比较其染色效果。结果丙酮固定的肝组织的免疫荧光组织结构清晰,核浆对比度好,其Heppar肝细胞抗原的阳性面积为(73.2±1.2)%,与10%甲醛(46.2±1.5)%及1%多聚甲醛(62.2±1.1)%固定的肝组织比较差异有统计学意义。结论在保存肝组织中抗原活性方面,相对于10%甲醛和1%多聚甲醛液,以丙酮固定液固定效果最好。

Exploration on method of tissue fixation for frozen sections stained with immunofluorescence

Objective To compare the effects of three different fixatives on frozen sections of liver tissue stained with immunofluorescence in order to find the best fixation way to preserve antigen.Methods The livers of Wistar rat were embedded with OCT and sectioned in freezing microtome,the thickness of sections were 8～10 μm.After fixed with with 10% formaldehyde,acetone and 1% paraformaldehyde respectively,the sections were stained with immunofluorescence,and image analysis were made by using the Image-pro image analysis software.Results The liver tissue fixed with acetone showed clear organizational structure and the nuclear plasma contrast was good,the Heppar antigen-positive area (73.2±1.2)%,which was higher than that in liver tissue fixed with 10% formaldehyde (46.2±1.5%)and 1% paraformaldehyde(62.2±1.1)%.Conclusion For purposes of antigen preservation,the fixed effect of acetone is batter than 10% formaldehyde and paraformaldehyde solution.