Lsm 800 microscope

To assess the enamel structural damage and mineral loss, we conducted multiscale surface analyses after removing the biofilms from the tooth enamel (24 (link), 39 (link)). In brief, after biofilm imaging, the biomass was removed using enzymatic treatment (dextranase and mutanase) followed by water-bath sonication, which was optimized for biofilm removal without causing artificial surface damage (24 (link)). Macroscopically, the demineralized areas on tooth-enamel surfaces (similar to those found clinically in severe childhood tooth decay) were visualized using stereomicroscopy (Zeiss AxioZoom v16). Then, the surface topography and roughness of the tooth-enamel surface were assessed by nondestructive confocal-topography using a 50× (numerical aperture = 0.95) objective on the Zeiss LSM800 microscope and ConfoMap software (24 (link)). Next, the tooth-enamel specimens were mounted on acrylic rods and sectioned (100 ± 20 μm thickness) with a hard-tissue microtome (Silverstone-Taylor Hard Tissue Microtome, Series 1000 Deluxe) for transverse microradiography. The sections were placed in the TMR-D system and X-rayed (45 kV, 45 mA) at a fixed distance for 12 s. An aluminum step wedge was X-rayed under identical conditions. The digital images were analyzed using TMR software (v3.0.0.18), with sound enamel defined at 87% mineral volume (51 (link)).

For the detection of intracellular ROS levels, 5 × 10⁵ vegetative D. discoideum cells were settled on glass plates for 30 min. A 10 μM 2′,7′-Dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich) was added to the cells and allowed to incubate for 30 min. Images (1 frame/min) of cells have been taken with a Zeiss LSM800 microscope with and without the addition of 1 μM Carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma- Aldrich) over a time period of 30 min. Alternatively, 5 × 10⁵ vegetative WT and mutant cells, washed and resuspended in PB, were seeded into wells of a black 96-wells-plate. A 10 μM DCF-DA was added to the wells and left to incubate for 30 min. Measurements (every minute) were taken immediately after the addition of 1 μM CCCP to the respective wells for 30 min via the microplate reader Synergy HTX (BioTek). Background measurements were taken of HL5 media with 10 μM DCF-DA and subtracted from the data. Measurements have been conducted five times.

Pieces of liver tissue were embedded in optimal cutting temperature (OCT) medium and kept at −80 °C until sectioning. Tissue blocks were cut into 10 µm sections and mounted onto Fisherbrand superfrost plus microscope slides. The sections were kept at −80 °C until analysis. OCT was removed by washing 2 × 5 min in phosphate buffered saline (PBS). Antigen retrieval was achieved by incubating sections in 1:10 Histo VT One solution at 65ºC for 40 min. The sections were blocked in 1% bovine serum albumin (BSA) and 0.1% Tween in 1 × PBS (PBST). After blocking, the sections were incubated in 1:200 rabbit anti-Atp7b antibody (EPR6794, Abcam) overnight at 4ºC. Sections were washed in PBST 4 × 5 min each. Secondary incubation was done in 1:500 donkey anti-rabbit 488 green antibody for 1 h. The sections were then washed twice with PBST and twice with PBS for 5 min each. Staining of nuclei was done by incubating slides for 5 min in 1:1000 Hoeschst 3342, trihydrochloride (Invitrogen) and then washing in PBS for 5 min. Sections were mounted using ProLong Gold antifade reagent and imaged with Zeiss LSM 800 microscope.

For apoptosis induction, cells were treated with 0.5 μmol·L⁻¹ STS (MedChemExpress, Shanghai, China) and incubated at 37 °C in 5% CO₂. After 12 h, cell supernatants were harvested and centrifuged at 300 × g for 15 min to remove cell debris. The supernatants were subsequently centrifuged at 3 000 × g for 20 min, and the pellets containing ABs were harvested for further experiments. For AB identification, the pellets were stained with Annexin-V/FITC and observed using a Zeiss LSM-800 microscope. For flow cytometry, isolated ABs were incubated with Annexin-V/FITC and PI in the dark for 15 min. After incubation, the samples were pelleted, the supernatant was removed, and the pellets were resuspended in 500 μL of PBS for flow cytometric analysis.

To stain OMVs, BV2 cells were grown on coverslips and infected with E. coli K1 for 2 h. After stimulation, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and blocked with 5% goat serum for 2 h prior to staining overnight at 4 °C with antibodies against LPS, followed by staining with fluorescently labeled secondary antibodies for 1 h and 1 μg/mL Hoechst-33258 for 20 min. Subsequently, the cells were sealed with a fluorescence quencher and visualized under a Zeiss LSM 800 microscope.