BMDMs, cultured on 8-chamber slides (Millicell® EZ SLIDES, MerckKGaA, Darmstadt, Germany) with different HP and treatments, were fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) for 30 min and blocked with 5% goat serum in 1x Perm Buffer (eBioscience) for 1 h at room temperature. Cells were then incubated with the primary antibody overnight at 4°C. After washing with PBS, the secondary antibody was added for 30 min. Cells were counterstained with Hoechst at 1:1,000 (B-2261; Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min at room temperature before embedding with moviol (Sigma-Aldrich) (27 (link)). The stainings were analyzed with a confocal microscope (LSM 710; Carl Zeiss, Jena, Germany) and images were acquired by ZEN acquisition software (2012; Carl Zeiss). For each marker, nine pictures from three independent experiments were randomly taken. The mean fluorescence intensity (MFI) and number of cells (based on nuclei within the viewing/visual field/field of vision) were quantified using ImageJ (1.80, National Institutes of Health, USA) (28 (link), 29 (link)).
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