固定剂
染色
溶解
污渍
细胞内
细菌细胞结构
抗原
化学
固定(群体遗传学)
多聚甲醛
活斑
戊二醛
抗体
免疫荧光
病理
微生物学
细菌
革兰氏染色
分子生物学
生物
生物化学
免疫学
遗传学
作者
Jian Sun,Yuan-Tian Mao,Lan-Yu Cui,Yongqiang Cao,Zhao Li,Min Ling,Xiao-Ping Xu,Shengbin He
出处
期刊:Methods and Applications in Fluorescence
[IOP Publishing]
日期:2021-04-26
卷期号:9 (3): 035001-035001
被引量:2
标识
DOI:10.1088/2050-6120/abf81e
摘要
The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2needed for 100% fixation is 50μg ml-1, which is much lower than that of traditional fixatives (1000-10000μg ml-1). The ClO2mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO2fixation on the staining. The results demonstrated that ClO2fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO2has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.
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