Plan apochromat 63x 1.40 oil objective

Chromosomes for 3D analysis measurements were prepared following the protocol of [46 (link)], using polyacrylamide pads. Confocal laser scanning microscopy was performed on inverted microscope Zeiss Axio Observer7 with laser scanning unit LSM880 equipped with four solid-state lasers (405 used), Plan-Apochromat 63x/1.40 oil objective, 32 channel spectral GaAsP detector, and Airyscan detector (increased resolution and signal to noise ratio). Images were captured using ZEN Black software, and 3D images were generated from Z-stack with fixed slice width and analysed using Imaris software (v. 9.8, Bitplane, Oxford Instruments, Abingdon-on-Thames, UK).

HEK293 cells transiently expressing POPDC1 and POPDC2 constructs tagged at the C-terminal with either enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) were incubated with 0.5% (v/v) CellBrite Red solution (Biotium) containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-dicarbocyanine (DiD) for 12-min at 37 °C to stain the plasma membrane. Cells were washed with PBS before fixation with PFA and stained with Hoechst-33342. Cells were imaged using a LSM 780 AxioObserver inverted confocal laser scanning microscope (Zeiss), with a plan-apochromat 63X/1.40 oil objective (Zeiss). Four channels: 405_ex/410–452_em nm, 458_ex/463–516_em nm, 514_ex/519–621_em nm and 633_ex/636–735_em nm, were used to image the Hoechst-33342, POPDC1-ECFP, POPDC2-EYFP and DiD, respectively. Cells were imaged in poly-L-lysine coated 8-well microscope slides with a D263 M Schott glass, No. 1.5H, 170 μm ± 5 μm glass coverslip base (Ibidi) immersed in PBS.

Purified monocytes were co-stained with the biotinylated NW peptide in combination with mouse anti-prohibitin monoclonal antibody in Eppendorf tubes. After incubation at 4 °C for 40 min, the cells were washed with FACS buffer and then incubated with PE-conjugated streptavidin and FITC-conjugated anti-mouse IgG for 30 min at 4 °C before being washed and resuspended in 50 μL of PBS buffer. A 20 μL drop of each sample was placed in the center of a glass slide and incubated at RT for 15 min. Thereafter, the samples were fixed with 4% paraformaldehyde, left for 20 min at RT, and washed with PBS. The slides were mounted with DAKO mounting medium and covered by cover slips for imaging with the Zeiss LSM 710 confocal microscope using the Plan-Apochromat 63x/1.40 oil objective (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Images were processed with the ZEN lite software (Carl Zeiss Microscopy GmbH, version 3.3).

Swim-up sperm cells were stained with the nuclear dye Hoechst 33342 while amyloid fibrils were stained with the amyloid-binding dye Proteostat or pFTAA (a luminescent-conjugated oligothiophene dye) as previously described (Usmani et al., 2014 (link)). A total of 10⁵ spermatozoa (final concentration 10⁷/ml) were mixed with 0, 2, 10, 50 and 250 µg/ml of the indicated synthetic amyloid fibril or purified endogenous amyloids (from the equivalent of 0.34, 0.17, or 0.8 ml SP, see details below) in a final volume of 50–100 µl, and then incubated at 37°C for 15–20 min. Images were acquired for 20 s with an interval of 1 s using a Plan-Apochromat 63X/1.40 oil objective lens on a LSM710 confocal microscope (Zeiss, Germany) equipped with Zen-Software (Zeiss). All experiments were performed at 37°C. A total of 3–5 experiments was performed for each donor to calculate the number of entrapped spermatozoa. Only live and motile spermatozoa were considered for analysis. The total number of free spermatozoa and spermatozoa trapped within the amyloid network was determined from all frames over a 20 s period and then the percentage of entrapped cells was calculated.

Laser-induced DNA damage induction was performed as in [50 ]. Following RNA silencing, ~ 80.000 U-2 OS cells were plated per well of a four-well Lab-Tek II chambered coverglass 24 h before imaging. Cells were imaged in FluoroBrite™ DMEM supplemented with 10% FBS, 25 mM HEPES, and sodium pyruvate. Laser induced laser stripes were done on a Zeiss LSM 800 microscope, using a 405 nm diode laser (5 mW) with the timed bleach option (60 iterations, 80% laser power output) in the ZenBlue 2.1 software using a Plan-Apochromat 63x/1.40 Oil objective after pre-sensitizing the cells with 1 μg/ml Hoechst 33342 (Molecular probes) for 30 min. 10 min after irradiation, cells were washed with ice-cold CSK extraction buffer (10 mM PIPES [pH = 7.0], 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl₂, 1 mM EGTA, 0.2% Triton X-100, cOmplete™ ULTRA protease inhibitor (Roche), PhosSTOP phosphatase inhibitor (Roche)) for 5 min and were subsequently fixed in 4% PFA in PBS for 15 min at room temperature. Samples were then blocked in blocking buffer (PBS containing 0.05% Triton X-100, 5% FBS and 3% BSA) before incubation with indicated primary then corresponding secondary antibodies (Supplemental Table S2). Slides were mounted in ProlongDiamond with DAPI (Molecular probes). Imaging was performed using a Zeiss LSM 800 microscope using a Plan-Apochromat 63x/1.40 Oil objective.

MoDCs were plated in 8-well µ-slides (Ibidi) coated with 0.01% poly-l-Lysine (Sigma) at a density of 2E + 05 cells per well in 250 µL of RPMI supplemented with 10% FBS and 2 mM l-glutamine. Cells were infected with STM-LT2 or STM-D23580 and incubated for 2 h prior to fixation as described above.
Cells were washed twice with PBS, fixed with 4% paraformaldehyde (PFA; EMS) for 15 min and permeabilised with 0.05% Triton-X100 in PBS (Sigma) twice for 1min. Cells were blocked overnight at 4 °C with 250 µL blocking solution (5% human serum (Sigma), 5% FBS) per well.
The following day, MoDCs were incubated with anti-Salmonella CSA-1 FITC-conjugated antibody (KPL, Goat polyclonal, 1:100) in 140 µL of blocking solution for 1 h at RT. Following two washes with PBS for 1 min each, cells were incubated with 200 µL PBS containing 1 µg/mL DAPI (Thermo Fisher) for 10 min at RT. Finally, 10 µL of VECTASHIELD Antifade Mounting Medium (H-100, Vector laboratories) were added to each well.
Images were acquired on a Zeiss LSM 880 inverted confocal microscope with a Plan-Apochromat 63x/1.40 Oil objective and processed using the Zeiss Zen software and assembled in Adobe illustrator.