Using a safe and effective fixative to improve the immunofluorescence staining of bacteria
| Autor: | Xiaoping Xu, Lanyu Cui, Min Ling, Yuantian Mao, Jian Sun, Yongqiang Cao, Zhao Li, Shengbin He |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Lysis
Fluorescent Antibody Technique Escherichia coli O157 Fixatives chemistry.chemical_compound Bacterial Proteins Antigen General Materials Science Instrumentation Spectroscopy Fixative Staining and Labeling biology Chemistry Green Chemistry Technology Oxides biology.organism_classification Atomic and Molecular Physics and Optics Staining Cross-Linking Reagents Biochemistry biology.protein Antibody Lysozyme Chlorine Compounds Intracellular Bacteria |
| Zdroj: | Methods and Applications in Fluorescence. 9:035001 |
| ISSN: | 2050-6120 |
| DOI: | 10.1088/2050-6120/abf81e |
| Popis: | 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 ClO2 needed for 100% fixation is 50 μg ml−1, which is much lower than that of traditional fixatives (1000–10000 μg ml−1). The ClO2 mediated 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 using E. coli O157: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 ClO2 fixation on the staining. The results demonstrated that ClO2 fixation 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, ClO2 has potential practical applications in immunofluorescence staining and fluorescence in situ hybridization for single bacteria/cell analysis. |
| Databáze: | OpenAIRE |
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