Sp8x confocal microscope

Prior to fixation, both control and decellularized ECM layers were washed in PBS twice. Samples were fixated with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, and blocked with 1% BSA in PBS. Samples were incubated for 1 h with collagen IV antibody (1:50, Millipore, Amsterdam, The Netherlands), EMILIN1 antibody (1:100; Sigma), or FBN1 antibody (1:200; Sigma). Samples were incubated with Alexa Fluor 488 donkey anti-goat IgG (1:100, Life Technologies) or Alexa Fluor 488 goat anti-rabbit IgG (1:100, Life Technologies), and Alexa Fluor 596 donkey anti-goat IgG (1:100; Life Technologies) or rhodamine-phalloidin (1:40; Life Technologies) in the dark for 1 h. Nuclei were counterstained with DAPI (1:5000) for 15 min. Washing with 0.05% Tween in PBS occurred after every antibody incubation. Samples were mounted on object slides using Mowiol. Imaging was performed using a Leica SP8X confocal microscope (Leica Microsystems, Amsterdam, The Netherlands) and LAS X software. Z-stacks were made to capture all fluorescent signal (5 a 6 Z-stacks/coverslip). Projections of the z-stack images were analyzed with ImageJ to calculate the area of EMILIN1, FBN1, collagen IV, and F-actin.

Live imaging of the cells treated with compound TAL3PYR was performed on the A549 cell line. Cells were seeded in Ibidi imaging cell chambers (Ibidi^®, Gräfelfing, Germany) in 500 μL of medium, 5 × 10⁴ cells/well, and wer eleft in the cell incubator for 48 h (37 °C, 5% CO₂). Subsequently, cells were treated with a 10 μM solution of the respective compound and left in the cell incubator for 90 min to allow the compound to enter the cells. After incubation, the medium was replaced with 500 μL of fresh medium. The influence of the compounds on A549 cells before and after photoactivation (λ_exc = 405 nm, λ_em = 450–550 nm) was visualised using a Leica SP8 X confocal microscope (Leica Microsystems, Wetzlar, Germany).

A. fumigatus spores were stained with fluorescein isothiocyanate (FITC, Sigma) for 30 min at 37°C while shaking. After three washes with PBS, 3x10⁵ spores were added onto fully confluent A549 monolayers in 24 well glass bottom plates. Following 4 hours of infection, the wells were washed two times with PBS to remove non-adherent spores, and the non-internalized spores were stained with 0.1 mg/ml of Calcofluor White in PBS (CFW; Sigma) for 5 min at 37°C. The wells were washed again twice with PBS, fixed for 15 min at 37°C using 5% formaldehyde and stored in PBS protected from light. Fluorescence images from 9 fields of view were captured in each well using a 1.5 pin hole and a 40 x objective of a Leica SP8x confocal microscope (Leica Microsystems, Germany). External CFW stained spore fluorescence was excited with a 405nm LED and collected on a HyD detector (410-450 nm) and all spore FITC fluorescence was excited with a 488nm Argon laser and collected on a HyD detector (495-570nm). The number of external and internal spores within the total population in each field of view of pixel size (388.26 µm x 388.26 µm) were counted manually in FIJI (Schindelin et al., 2012 (link)) for all strains and expressed as % spores internalized. The % uptake relative to the parental strain was calculated of each strain.

Mitochondrial superoxide detection was performed by incubating the hiPSC-derived-neurons in HBSS containing 2.5 μM MitoSox Red mitochondrial superoxide indicator (Thermo Fisher Scientific) for 15 min at 37°C and 5% CO₂. After incubation, cells were washed three times with HBSS. Mitochondrial membrane potential was measured using 50 nM TMRM mitochondrial membrane potential indicator (Thermo Fisher Scientific) for 30 min at 37°C and 5% CO₂. Mitochondrial morphology was determined by staining the cells with 1 μM MitoTracker Green FM (Thermo Fisher Scientific) for 15 min at 37°C and 5% CO₂. Images were acquired in live using a Leica SP8-X confocal microscope (Leica Microsystems) and analyzed using Imaris software (Oxford Instruments).

For immunocytochemical analysis, differentiated neurons were grown on a 16-well format chamber slide System Nunc™ Lab-Tek™ (Thermo Fisher Scientific), previously coated with poly-d-lysine (Sigma Aldrich) and laminin (Sigma Aldrich), and fixed in a 4% paraformaldehyde solution. Next, cells were permeabilized in 0.5% Triton X-100—PBS and blocked with a blocking solution (3% BSA in PBS) at RT. Immunostaining was performed by the addition of primary antibodies to co-stain the DA neuronal marker TH (rabbit anti-TH, Merck Millipore MAB318) and the class III member of the beta-tubulin family (mouse anti-TUJ1, Biolegend). Subsequently, cells were incubated with the appropriate fluorescently labeled secondary antibodies (goat anti-mouse Alexa Fluor 488-conjugated, goat anti-rabbit Alexa Fluor 555-conjugated, Thermo Fisher Scientific), nuclei were stained by adding NucBlue™ Fixed Cell Stain (Thermo Fisher Scientific), and the slides were mounted with Dako Fluorescence mounting medium. Images were acquired using a Leica SP8-X confocal microscope (Leica Microsystems, LAS-X acquisition software), with hybrid detectors and a 63X oil immersion objective.

The Live/Dead cell viability assay (Live/Dead cell viability assay kit, Thermo Fisher Scientific) was performed at days 5, 10, and 20 of differentiation. Single beads were placed in a live imaging chamber and incubated for 20 min with a 2 μM Ethidium Homodimer-1 (EH-1) and 8 μM Calcein (AM) solution in PBS in dark conditions. Images were acquired using a Leica SP8-X confocal microscope (Leica Microsystems) and analyzed using Imaris software (Oxford Instruments).