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Immunofluorescence (IF) is widely used to study the cellular localization and organization of proteins. However, steps such as fixation and permeabilization may affect cell morphology and/or introduce artifacts. For bacterial cells, commonly used permeabilization methods for IF include treatment with lysozyme. Here, we demonstrate two potential pitfalls in IF due to specific permeabilization methods: flattening or disruption of the cells caused by lysozyme treatment and inaccessibility of the antibody to the fixed nucleoid region. To solve these issues, we propose an improved IF method for bacterial cells, which includes the combined treatment with 70% ethanol, lysozyme, and DNase I. Treatment with 70% ethanol before the lysozyme permeabilization can better preserve the three-dimensional shape of the cell, and treatment with DNase I after the lysozyme permeabilization can eliminate the inaccessibility of the antibody to the nucleoid region. We further demonstrate that the DNase I treatment does not affect the preservation of the DNA-associated structure or organization of proteins. Finally, the method is also compatible with applications in which IF needs to be combined with RNA fluorescence in situ hybridization.

An Improved Method for Bacterial Immunofluorescence Staining To Eliminate Antibody Exclusion from the Fixed Nucleoid