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.

中文翻译：

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使用安全有效的固定剂来改善细菌的免疫荧光染色。

绿色化学的兴起和发展再次吸引了研究人员的注意力，以消除有害物质的使用和产生。在这里，我们报告了使用安全有效的固定剂二氧化氯（ClO2）代替传统的危险固定剂来使细胞蛋白交联，以改善细菌的免疫荧光染色。100％固色所需的ClO2浓度为50μgml-1，远低于传统固色剂（1000-10000μgml-1）。ClO2介导的交联可以保留细菌细胞的完整性，并防止细胞因裂解而损失。同时，溶菌酶可以渗透细菌细胞，使标记的抗体扩散到其细胞内靶分子。通过使用E。coliO157：H7 / RP4作为革兰氏阴性细菌模型，进行了细胞内蛋白和表面多糖的免疫荧光染色试验，以研究ClO2固定对染色的影响。结果表明，ClO2的固定可以防止靶抗原裂解细菌，而不会破坏抗体和抗原（无论是多糖还是蛋白质）之间的相互作用。作为一种安全有效的固定剂，ClO2在免疫荧光染色和荧光原位杂交中的单个细菌/细胞分析中具有潜在的实际应用。结果表明，ClO2的固定可以防止靶抗原裂解细菌，而不会破坏抗体和抗原（无论是多糖还是蛋白质）之间的相互作用。作为一种安全有效的固定剂，ClO2在免疫荧光染色和荧光原位杂交中的单个细菌/细胞分析中具有潜在的实际应用。结果表明，ClO2的固定可以防止靶抗原裂解细菌，而不会破坏抗体和抗原（无论是多糖还是蛋白质）之间的相互作用。作为一种安全有效的固定剂，ClO2在免疫荧光染色和荧光原位杂交中的单个细菌/细胞分析中具有潜在的实际应用。