Mounting medium

Cells were grown on 4-, 8- or 18-well µ-slides (ibidi, Gräfelfing, Germany) to the corresponding stages, rinsed twice in PBS pH 7.4 and fixed in 4% paraformaldehyde in PBS for 15 min, washed 3x in PBS for 5 min, permeabilized in 0.5% Triton X-100/PBS for 15 min and washed 3x in PBS for 5 min, all at room temperature. Cells were pre-incubated in 5% normal donkey serum (NDS) in permeabilization solution for 1 h at room temperature and incubated with primary antibodies (Table 3) overnight at 4 °C. After 3 × 10 min washes in PBS, cells were incubated with Alexa Fluor 488 and 555 secondary antibodies (Table 4) in 2.5% NDS in permeabilization solution for 1–2 h, washed 3 × 10 min with TBS (50 mM Tris-HCl pH 7.5; 0.9% NaCl) and incubated with 0.5 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 15 min, all at room temperature. After 3 × 5 min washes in TBS, cells were mounted in mounting medium (ibidi) and imaged on a DMi8 widefield microscope (Leica Microsystems, Wetzlar, Germany). Images were acquired with a DFC9000 GT sCMOS camera using LAS X Premium software (Leica Microsystems) and processed with Adobe Photoshop (Adobe, San Jose, CA, USA) software.

The larvae were washed and subsequently stained with Nile Red (Sigma, Jerusalem, Israel), which specifically stained neutral lipids, such as triglycerides [64 (link)]. The larvae were exposed to Nile Red (500 ng/mL) for 0.5 h and subsequently euthanized. Using a 1% low melting point agarose or a glycerol-based Mounting Medium (ibidi Mounting Medium, ibidi, Grafelfing, Germany), each larva was positioned on its right side (location of adipocyte deposit) on a coverslip assembly, based on a published diagram [65 ] (wiki.zfin.org/display/prot/Viewing+Chambers, last accessed on 2 July 2023). The degree of obesity was assessed by the number of Nile Red-stained lipid droplets (assumed to be in adipocytes) and their area, as determined by confocal microscopy (Zeiss LSM 700 laser scanning confocal microscope, using an X5 objective, laser settings of 488 nm for excitation and ≥539 nm for emission and ZEN 2.6 (blue edition) software (Zeiss)). Conditions, including the laser power, pinhole, master gain, and digital offset were kept constant for each experiment. The layer counted was the one with the most Nile Red-stained lipid droplets in the abdominal area. Lipid droplets smaller than 100 µm² were excluded, as were those appearing in a row (since they could be fat droplets in lymph vessels and not adipocytes).

The preparation of samples for observations of antheridium by light and fluorescent microscopy was performed as described previously^{27 (link)}. Each specimen was stained with DAPI (0.25 μg/ml) and mounted in ibidi mounting medium (ibidi, Germany). Microphotographs were taken using a BX 51 microscope (Olympus, Tokyo, Japan) equipped with a DP70 digital camera (Olympus).

For detection of viral antigens, ASK cells were fixed in acetone (80%, 10 min, RT) or paraformaldehyde (4%, 10 min, RT), washed, incubated with primary antibodies (60 min, RT), washed three times in PBS, incubated with goat anti-mouse IgG–Alexa 488 (Thermo Fisher Scientific, 45 min, RT), washed three times in PBS, counterstained with Hoechst 33342 (Sigma-Aldrich, 2 μg/mL, 3 min, RT), and stored and imaged in PBS or mounting medium (Ibidi GmbH). Specific antibodies and dilutions are given in S1 Table. For immunofluorescent staining of tissues, deparaffinized and heat treated (60–70°C, 20 min) sections of formalin-fixed paraffin-embedded tissues were incubated with mouse monoclonal IgM targeting an epitope closely associated with the ISAV-binding epitope (10E4 [20 (link)], 1:100, RT, 60 min), washed in PBS, and incubated with goat anti-mouse IgM—Alexa594 (Thermo Fisher Scientific, 45 min, RT). Wide-field imaging was performed on a Zeiss Axio Observer A1 fluorescent microscope with a 40x LD-plan Neofluar 40x objective (N/A 0.6) or a Plan Neofluar 40x oil objective (N/A 1.3). Confocal imaging was performed on a Zeiss LSM710 confocal microscope with Plan Apochromat SF25 40x oil objective (N/A 1.3) or a Plan Apochromat 63x oil objective (N/A 1.4).

Cell samples were washed with PBS and fixed in 3.7% paraformaldehyde/PBS solution. For immunolabeling, cells were permeabilized with 0.3% Triton X-100 and 0.1% BSA in PBS. Autofluorescence was quenched with 50 mM NH₄Cl in PBS. Primary antibodies were applied in 16% goat serum, 0.3% Triton X-100, 0.3 M NaCl in PBS, and incubated overnight. Subsequently, cells were washed three times with 0.3% Triton X-100 and 0.1% BSA in PBS at room temperature. Secondary antibodies were applied in same buffer and incubated for one hour. For ThioflavinS (ThS) or phalloidinalexa647 staining, fixed cells were incubated with 0.05% ThS or 70 nM phalloidinalexa647 in PBS for 15 minutes. Nuclear counterstaining was performed by incubation in 300 nM 4',6-diamidino-2-phenylindole (DAPI) in PBS for 10 minutes. After washing with PBS, samples were mounted with mounting medium (Ibidi, Germany).

Eosinophils primed with either IL3 or IL5 (both at 2 ng/mL) for 20 h, treated with bafilomycin-A1 (1 μM) or vehicle alone for 20 min, were seeded at 0.5 × 10⁶ cells/mL (300 μL/well) in an eight-well chamber slide (Ibidi, Gräfelfing, Germany) coated with 300 μL of HA-IgG (10 μg/mL). After 4.5 h, cells were washed and fixed with 4% paraformaldehyde for 20 min at room temperature (RT). After three washes with PBS, DAPI was added into wells for 10 min at RT, followed by three washes and the addition of mounting medium (Ibidi). Images were taken using a Nikon Eclipse Ti microscope and its 20× objective. Bright-field (BF) images and images of DAPI fluorescence (200 msec. exposure) were taken using the BF and DAPI settings on the NIS-Elements AR v4.30.02 software. BF images allowed cell counting while DAPI fluorescence was quantified by ImageJ (https://imagej.nih.gov/ij/ accessed on 23 April 2018) (threshold fixed around 6500). For each well, three to four fields (depending on the cell density in the field) were randomly used for a total cell count of ~450 cells. The total area of DAPI fluorescence per cell was calculated, and the number of DNA projections per 100 cells was counted. A projection was defined as a line of DNA with a length >2-fold the length of an intact nucleus.