用于甲醛固定石蜡包埋组织的改良免疫荧光法

摘要

BACKGROUND: Formalin fixed paraffin embedded tissue (FFPE) is apt for storage, and immunofluorescence method supportsprecise localization and quantitation as well as multiple labeling.OBJECTIVE: To establish an upgraded immunofluorescence method for FFPE tissue.METHODS: Paraffin sections of synovial vascular mantle and acetabular hyperplasia were obtained from rheumatoid arthritis andosteoarthritis patients and then preformed with routine dewaxing and rehydration. The retrieve durations, serum blockage time andprimary antibody concentrations of different citric acid buffer and the color difference of fluorescent markers in theimmunofluorescence were observed.RESULTS AND CONCLUSION: Compared with simple high-temperature repairing for 10-20 minutes, the tissue structure waspreserved completely after high-temperature repairing for 15 minutes with citric acid followed by antigen retrieval solution, andthere was no significant difference in background staining; the blockage of normal serum with 10% concentration of secondantibody under room temperature was regarded as the best conditions of antigen. Under this condition, we could guarantee a goodsealing effect, and would not affect the binding of primary antibody and antigen sites. Higher concentration of primary antibody(10 mg/L) could ensure a clear fluorescence signal, low background and acceptable cost. The paraffin sections were preformedwith immunofluorescence assay, and observed with an ordinary fluorescence microscope. The FITC excitation light blue and greenfluorescence signal marked dye groups were avoided. High-temperature repairing for 15 minutes with citric acid followed byantigen retrieval solution, close the normal serum with 10% concentration of second antibody under room temperature for40 minutes, relatively high primary antibody concentration and observe under ordinary fluorescence microscope can improve thequality of immunofluorescence detection.%背景:甲醛固定石蜡包埋的标本易于保存,免疫荧光法定位定量能精确支持多种标记物.目的:建立适合于甲醛固定石蜡包埋组织的改良免疫荧光法.方法:将取自类风湿性关节炎和无骨性关节炎患者的滑膜血管翕和髋臼增生组织的石蜡切片进行常规脱蜡复水,观察不同的柠檬酸缓冲液热修复时间、血清封闭时间、一抗浓度和荧光标记物在免疫荧光法中显色的差异.结果与结论:柠檬酸高温修复15 min 后滴加抗原修复液比单纯高温修复10～20 min,组织结构保存更为完整,且背景染色无显著区别;选择体积分数10%二抗来源正常血清室温封闭40 min 作为最佳的抗原封闭条件,既可以保证良好的封闭效果,同时又不影响一抗和抗原位点结合;较高一抗浓度(10 mg/L) 能保证明显的荧光信号、低背景和可接受的成本.进行石蜡切片免疫荧光实验,并用普通荧光显微镜观察,应避免采用FITC 等蓝色激发光、呈现绿色荧光信号的标记染料基团.提示柠檬酸高温修复15 min 后滴加抗原修复液,选择体积分数10% 二抗来源正常血清室温封闭40 min,较高一抗浓度,普通荧光显微镜观察可提高免疫荧光法的检测质量.