摘要
目的以氯通道蛋白ClC-3为靶蛋白,建立一种不溶血的红细胞免疫荧光细胞化学检测方法。方法采集大鼠红细胞,涂片,0.5%丙烯醛/PBS固定,以不同浓度的Triton X-100/PBS(含0.1 mol/L甘氨酸)溶液透膜,用含0.05 mmol/L甘氨酸、2%BSA、0.05%NaN3的PBS溶液封闭抗原,随后孵育ClC-3的一抗和荧光标记的二抗,最后在共聚焦荧光显微镜下观察结果。结果 0.1%Triton X-100/PBS结合其他优化液处理的红细胞未出现溶血现象,共聚焦显微镜下清晰地观察到目的蛋白及其亚细胞分布情况。结论通过优化固定液、透膜液、封闭液和洗涤液,解决了常规免疫细胞化学法导致红细胞易溶血的缺点,在确保红细胞不溶血的同时成功检测到红细胞氯通道ClC-3蛋白,即成功建立了一种不溶血的红细胞免疫荧光细胞化学检测方法。
Objective To establish an immunofluorescence cytochemical method utilizing the immunofluorescence technique for detecting the protein of the RBC by taking the chloride channel protein ClC-3 as the target protein.Methods Rat RBCs were collected,smeared and then fixed with 0.5％ acrolein solution and permeabilized in PBS containing 0.1 M glycine plus 0.01％ Triton X-100.Nonspecific binding was blocked by incubation in blocking buffer containing 0.05 mM Gly,2％ BSA and 0.05％ NaN3.After the incubation of primary and fluorescence-linked second antibody,detection of labeled samples was performed with a confocal laser-scanning microscope.Results RBCs maintained the typical red double-concave disc structure,and there was no hemolysis.Clear ClC-3 protein expression and distribution was observed.Conclusions We conclude that the method resolves erythrocytes hemolysis problem easily in immunofluorescence experiment and proves to be an efficient method to detect the expression of RBCs protein.Therefore,we establish an immunofluorescence cytochemical detection method successfully.
出处
《山东医药》
CAS
2013年第43期24-26,共3页
Shandong Medical Journal
基金
国家自然科学基金资助项目(31371144)
关键词
红细胞
免疫荧光技术
免疫荧光细胞化学检测方法
氯通道蛋白
溶血
erythrocytes
immunofluorescence technique
immunofluorescence cytochemical method
chloride channel protein
hemolysis
