Prolong gold mounting solution

C2C12 myoblasts stably expressing mCherry‐GFP‐mtFIS1_101‐152 were seeded on imaging dishes (Ibidi, Gräfelfing, Germany) and treated with either 10 μM of CCCP or 20 μM of AMPK activator 991. Following treatment, cells were washed twice with PBS and fixed in 3.7% of formaldehyde with 200 mM of HEPES (pH 7.0) for 10 minutes. After fixing, cells were washed and incubated for 10 minutes in DMEM supplemented with 10 mM of HEPES (pH 7.0), and then washed with PBS before mounting with Prolong gold mounting solution containing 4′,6‐diamidino‐2‐phenylindole (DAPI; ThermoFisher Scientific, Leicestershire UK). Images were taken using a Crest X‐Light spinning disk system coupled to a Nikon Ti‐E base, 60x/1.4 NA (CFI Plan Apo Lambda) air objective and Photometrics Delta Evolve EM‐CCD. For GFP, excitation was delivered at λ = 458‐482 nm using a Lumencor Spectra X light engine, with emitted signals detected at λ = 500‐550 nm. For mCherry, the wavelengths used for excitation and detection were λ = 563‐587 nm and λ = 602‐662 nm, respectively.

Cell cycle progression was examined using the Click-it EdU Alexa Fluor 647 kit (Thermo Scientific). Briefly, cells were plated on cover slips. After 24 hours, cells were pulsed in EdU for 10 minutes and then fixed in 4% paraformaldehyde in PBS, permeabilized in 0.1% TritonX in PBS. Cells were then washed twice in 2% BSA in PBS. The EdU was reacted with azide-conjugated fluorophore in the presence of copper sulfate. The click chemistry reaction was allowed to proceed for 1 hour. Cells were washed twice in 2% BSA in PBS. DNA was labeled using Hoechst at a dilution of 1:10,000 mounted on Prolong Gold mounting solution (Thermo Scientific). The fraction of cells in each stage of the cell cycle was quantified using a previously published MATLAB (The MathWorks, Natick, MA, USA) analysis pipeline [66 (link)] and is available at https://github.com/scappell/Cell_tracking.

BMM cells were incubated with 1000 ng/mL isolated RANKL in four‐well chamber slide. After 1 h, the cells were fixed in 4% paraformaldehyde. Then, the cells were incubated with rabbit anti‐NFATc1 (1:200, diluted in PBST containing 5% BSA) for 2 h. After washing with PBST, the cells were incubated with FITC‐labeled goat anti‐rabbit IgG antibody (10 μg/mL diluted in PBST containing 5% BSA) for 1 h in the dark and washed with PBST for 10 min. Immunolabeled cells were counter‐stained with 4′,6′‑diamidino‑2‑phenylindole (DAPI) contained in Pro‐Long Gold mounting solution (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Digital images were acquired at the Korea Basic Science Institute Gwangju Center using a TCS SP5 AOBS (Leica Microsystems, Heidelberg, Germany) laser‑scanning microscope.

Cells were rinsed with PBS and immediately fixed using 4% paraformaldehyde with 4% sucrose in PBS for 15 min. After rinsing with PBS, a 15-min permeabilization using 0.2% Triton X-100 in PBS, and a 1-h blocking solution (4% BSA and 4% normal goat serum in PBS), cells were incubated overnight at 4°C in primary antibody diluted in blocking solution (mouse anti-CaMKII from Millipore 05-532, rabbit anti-Gluc from Nanolight 401P, mouse anti-GAD67 from Millipore MAB5406, mouse anti-GFAP from Cell Signaling Tech. 3670, chicken anti-MAP2 from Abcam ab5392). After washing with PBS, the cells were incubated with a fluorescent secondary antibody and Hoeschst (Hoechest 33342 from Invitrogen, donkey anti-rabbit Alexa Fluor 488 from Invitrogen A21206, all others from Jackson ImmunoResearch). Coverslips were mounted onto glass slides using Prolong gold mounting solution (Invitrogen P36930). Imaging was performed using either a Nikon E800 epi-fluorescence microscope or Nikon A1 for confocal imaging.

iDCs (3 × 10⁵ cells) were transferred to wells of a 24-well plate containing polylysine-coated coverslips (12 mm diameter) and FITC-labelled conidia of A. fumigatus or A. terreus were added at a multiplicity of infection (MOI) of 0.5. At selected time points plates were centrifuged at 200 × g to pellet cells and fixed over night at 4 °C in the presence of 4% of paraformaldehyde. After washing with PBS, cells were blocked for 15 min at room temperature (RT) with Fc receptor blocking solution (Human TruStain FcX^TM, BioLegend). Plates were centrifuged again for 10 min at 200 × g before blocking solution was removed. To stain conidia not phagocytosed by DCs, a rabbit anti-conidia antiserum (kindly provided by Frank Ebel, Munich) diluted 1:100 in 1% bovine serum albumin (BSA, sigma) was added and incubated for 1 hour at RT. After washing with PBS, Cy3-labelled anti-rabbit IgG antibody diluted 1:300 in 1% BSA was added and incubated for 1 hour at RT in the dark. Coverslips were washed three times with PBS, mounted in ProLong Gold mounting solution (Invitrogen) and analysed by fluorescence microscopy. Conidia co-localising with DCs were counted. Phagocytosis rates were calculated from FITC⁺, Cy3⁻ conidia divided by the total number of conidia counted resulting in the percentage of phagocytosis. At least 900 conidia were analysed in each experimental group.

Whole-mount brains were mounted on a 76 × 26 mm glass microscope slide (KnittelGlass) to which pairs of Paper Reinforcement Rings had been applied. The samples were mounted in Prolong Gold mounting solution (Invitrogen). Spacers were covered with a cover glass (KnittelGlass, #1 thickness, 0.17 mm) held in place by nail polish. Image acquisition was done sequentially on a Leica TCS SP8 upright microscope with a 25 × 0.95 NA plan-apochromat water immersion objective. The original image data consisted of 1, 024 × 1, 024 × ~200 voxels, with a voxel size of 0.60 × 0.60 × 0.98 μm. Images were acquired with a 12-bit dynamic range. A frame average of two successive scans was applied. Fluorescence emission from the 488, 547, and 647 nm was imaged using the 488, 561, and 633 nm lasers, respectively. The laser power was increased along the z-axis to compensate for signal attenuation.