原位细胞凋亡TUNEL法的改进及其应用

Improved method for TUNEL technique and its applications

目的 :寻找一种能快速、准确检测以石蜡切片、冰冻切片与细胞爬片或涂片等为实验材料的原位细胞凋亡的 TUNEL法。方法 :在原已建立的 TUNEL技术基础上作了如下改进 :1石蜡切片用微波修复技术替代蛋白酶 K的消化作用 ;冰冻切片与细胞爬片或涂片用含 0 .1 % Triton X- 1 0 0和 0 .1 %枸橼酸三钠混合液作穿透处理。 2在滴加反应混合物前 ,切片用 0 .1 %焦碳酸二乙酯 ( DEPC)浸泡 ,抑制内源性核酸内切酶活性。 3在 POD转换剂前 ,切片用 3% H2 O2 -甲醇液阻断内源性过氧化物酶活性 ,并用正常山羊血清封闭非特异结合位点。4苏木素套染细胞核。结果 :改进后的 TUNEL法能特异地显示凋亡细胞为棕黄色 ,其他细胞核为蓝色 ,且对比明显 ,背景浅 ,组织结构完整清晰 ,便于显微照相等特点。结论 :改进后的 TUNEL法是一种快速、准确检测以石蜡切片、冰冻切片与细胞爬片或涂片等为实验材料细胞凋亡的有效方法。

Objective: To look for a rapid,precise and effective method to detect in situ apoptosis in paraffin-embedded sections, frozen tissue sections, adherent cells and smears. Methods:The TUNEL technique established previously was improved as follow: ① Microwave retrieval technique was used instead of proteinase K to pretreat the paraffin embedded sections; Frozen tissue sections, adherent cells and smears were permeated with 0.1% Triton X-100 in 0.1% sodium citrate. ② The sections were immersed in 0.1% diethyl-pyrocarbonate (DEPC) to inhibit the activity of endogenous endonuclease before the TUNEL reaction mixture was added. ③ The activity of endoperoxidase was blocked by 3% H2O2 in methanol and nonspecific sites were blocked up with normal sheep serum before the converter-POD was added. ④ Counterstained by hematoxylin. Results:The improved method for TUNEL could specifically show apoptotic nuclei yellow and the others blue; it revealed the sharper characteristic contrast, lighter background, more detail and clearer structure of tissues and cells,and was suitable for microphotography. Conclusion:The improved method for TUNEL is a rapid,precise and effective method to detect apoptosis in paraffin-embedded or frozen sections, adherent cells and smears, etc.