Ab5605

Fibroblasts were grown on coverslips at a density of 1 × 105 cells/mL, after 24 h of treatment with plain ETHO and ETHO-CoQ10, cells were exposed to 50 μM H₂O₂ for 1 h. After H₂O₂ exposure, the medium was changed with fresh medium and the cells were analyzed at three different time points (i.e., directly post exposure, 2 h and 6 h post-exposure). Afterwards, cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT). Cells were then permeabilized for 5 min at RT with PBS containing 0.2% Triton X-100, then the coverslips were blocked in PBS containing 1% BSA at RT for 1 h. Cells were then incubated with primary antibody for 4HNE (AB5605; Millipore Corporation, Burlington, MA, USA) (1.200) in PBS containing 0.5% BSA at 4 °C overnight. After washing, coverslips were incubated with appropriate secondary antibody (1:100) for 1 h at RT. Nuclei were stained with 1 μg/mL DAPI (Sigma-Aldrich, Merck, Darmstadt, Germany) for 1 min. Coverslips were mounted onto glass slides using anti-fade mounting medium 1,4 diazabicyclooctane (DABCO) in glycerin and examined by the Leica light microscope equipped with epifluorescence at 40× magnification. Negative controls for the immunostaining experiments were performed by omitting primary antibody. Images were acquired and analyzed with Leica software [41 (link)].

Total cell counts in BAL was measured with the Coulter Counter (Beckman Coulter, Brea, CA, USA). A sample containing 3 × 10⁴ BAL cells was added to cytospins (Shandon cytospin 3, Thermo Scientific, Rockford, IL, USA) and were manually differentiated by counting 400 cells in a blinded fashion. The following cytokines were measured in BAL by magnetic Luminex assay (R&D, Minneapolis, MN, USA): IFN-y, KC, MIP-1α, IL10, IL17A, IL6. Lung permeability was determined by measuring the high-molecular-weight protein IgM in BAL, a large protein that is absent in BAL of non-infected mice, by ELISA (Bethyl Laboratories, Montgomery, TX, USA), as described before [25 ]. BAL neutrophil degranulation was measured by myeloperoxidase (MPO) ELISA (DY3667, R&D). ROS-production was measured by quantitative western blot for detection of 4-Hydroxynenal (4HNE, AB5605, Millipore, Darmstadt, Germany) in BAL normalized for protein content by Bradford assay. 4HNE is produced under influence of ROS [26 (link)].

Paraffin-embedded 4 µm sections of skin biopsies were deparaffinized in xylene and rehydrated in decreasing alcohol gradients. Antigen retrieval was achieved using heat-based epitope retrieval with sodium citrate buffer (Thermo Fisher Scientific, Waltham, MA, USA) (pH 6.0) at a sub-boiling temperature in a 500-watt microwave for 10 min. After cooling, sections were washed in PBS, blocked with 5% BSA in PBS, and incubated with primary antibodies for 4HNE (dil. 1:400; AB5605, Millipore), MMP9 (dil. 1:200; NBP2-13173, Novus Biologicals), filaggrin (dil. 1:50; sc-66192, Santa Cruz Biotechnology, Inc.), in 2% BSA in PBS. Sections were then washed in PBS and incubated with fluorochrome-conjugated secondary antibodies (dil. 1:1,000) (Alexa Fluor 568 A11004 or Alexa Fluor 488 A11055) in 2% BSA in PBS at RT, and then washed with PBS. Nuclei were stained with DAPI (1874814, Invitrogen) in PBS, and sections were then washed with PBS. Sections were mounted using PermaFluor mounting medium (Thermo Fisher Scientific) and imaged on a Zeiss LSM10 microscope. Images were quantified using ImageJ.