Gold antifade reagent

Sperm samples were washed with PBS and then diluted to 1 × 10⁷/ml. 30% H₂O₂ were added in the volume of 1 μl, 5 μl and 10 μl to the total of 1.5 ml sample and then were treated for 3 hours before analysis with Western blot.
For immunostaining, 1 ml of sperm suspension was centrifuged at 1,000 rpm for 6 min, the sediment was incubated in 100 μl decondensation buffer (2.5 mM DTT, 0.2% Triton-X-100 in PBS) for 20 min, then incubated in 0.5% (v/v) heparin in decondensation buffer for 4 min. 10 μl of the decondensated sperm suspension was brought onto a glass slide and spread out with the pipet tip, then air dried. The slide was subsequently incubated in 4% PFA for 15 min, air dried, washed in photo-fluo diluted 1:200 in PBS. After drying, the slide was washed in PBS-T twice, permeabilized with 0.2% Triton-X-100 in PBS for 15 min and blocked in 5% BSA/PBS for 1 h at room temperature, followed by incubation of primary antibody diluted 1:200 in blocking solution overnight at 4 °C. The slide was washed in PBS-T three times for 15 min, then incubated in secondary antibody diluted 1:500 in blocking solution in dark for 1 h at room temperature and washed three times in PBS-T. After then the slide was incubated in 2 μg/ml DAPI for 8 min, washed once and mounted with Gold anti-fade reagent (Life Technologies).

5 x 10⁵ (300 μl/well) tonsil-derived cells were seeded on a poly-L-lysine-coated coverslip in a 24-well plate. Cells were incubated for 2 h at 37°C or 4°C with or without heat-inactivated S. aureus or PhRodo Green S. aureus Bioparticles at a MOI of 10:1 (bacteria:cells). After incubation, cells were fixed with 2% formaldehyde for 30 min at RT, washed with PBS and permeabilized with 0.1% Triton X-100. Coverslips were then washed again with PBS and blocked with 3% BSA—10% goat serum (Invitrogen) for 20 min at RT. Finally, coverslips were washed with PBS and incubated for 30 min at RT with antibody mix containing human anti-CD19-V450 HIB19 (BD Pharmingen), anti-GM-CSF-eFluor660 GM2F3, anti-MPO-PE 455-8E6 (eBioscience), all diluted in 0.1% BSA + 1% goat serum. After incubation, coverslips were washed and mounted on a slide with Gold Anti-Fade reagent (Life Technologies) for analysis by confocal microscopy.

After following standard histologic procedures, sections were deparaffinized in xylene and rehydrated by a graded ethanol series (100% × 5 min, 85% × 5 min, 75% × 5 min, and Milli-Q H₂O × 5 min). One drop of the PNA probe in the hybridization solution, including a FAM-labeled PNA probe (5′-FAM-GAAGCAAGCTTCTCGTCCG-FAM-3′) targeting S. aureus 16S rRNA (Servicebio, Wuhan, China), was applied to the rehydrated bone. A coverslip was applied, and the slides were incubated for 90 min at 56 °C. The coverslips were removed, and the slides were incubated for 30 min in the wash buffer (AdvanDx, Woburn, MA, USA) at 56 °C. The slides were removed and dried in the air. The bone sections were covered in approximately 100 μL of 4,6-diamidino-2-phenylindole (DAPI; ThermoFisher, Waltham, MA, USA) and incubated in the dark for 30 min at room temperature. The DAPI solution was washed off with phosphate-buffered saline (PBS), and the samples were allowed to dry in the air. One drop of Gold Antifade Reagent (Thermo Fisher, Waltham, MA, USA) was added to each section. A coverslip was applied, and the samples were incubated overnight in darkness. The coverslips were then sealed with clear nail polish, and the slides were then stored at 4 °C.

The NIH3T3 Cells and L929 cells were fixed with paraformaldehyde, treated with blocking buffer (0.2% sodium azide, 2% bovine serum albumin, and 0.1% Triton X-100 in TBS-Tween) for 1 h, and incubated with the primary antibodies at 4 °C overnight. The primary antibodies were diluted as follows: NRF2 (1:1000, Novoprotein, Suzhou, Jiangsu, China), Ki67 (1:1000, Proteintech, Chicago, IL, USA). Furthermore, the cellular nuclei were stained with DAPI (1:500, Sigma, Aldrich, MO, USA) for 8 min. Finally, slips were mounted in gold antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA) and captured on a confocal microscope (Olympus, Tokyo, Japan).