Axio imager a2

The sexual structures were observed under a light microscope Axio Imager A2 ( Zeiss, Jena, Germany) and a stereo microscope SZX2-ILLT (Olympus, Tokyo, Japan). The structures were also paraffin sectioned or stained with calcofluor white (50 μg/mL) and detected under a fluorescence microscope Axio Imager A2 (Zeiss, Jena, Germany) or a laser scanning confocal fluorescence microscope LSM880 (Zeiss, Jena, Germany). Cryoelectronic scanning electron microscopy Regulus 8100 (Hitachi, Tokyo, Japan) and transmission electron microscopy H7650 (Hitachi, Tokyo, Japan) were also used to analyze the ultrastructure of the sexual offspring.

Cells were seeded in coverslips dishes at a density of l × 10⁴ cells/cm², maintained 24 h for attachment and then treated or not with BSHE (whose GR50 concentrations were those referred to the 48 h-exposure). After 48 h-incubation, cells were washed twice with PBS, stained with 4 μg/mL AO for 10 min and washed again with PBS. The coverslips were overturned on microscope slides and immediately observed under fluorescence microscopy (Axio Imager A2 Zeiss). Images were acquired using a color camera (DFC320 Leica). AO is a metachromatic dye whose luminescence wavelength is strongly dependent on its concentration. In live cells, AO is accumulated by acidic vesicles yielding strong orange or red signals; it is also provided with affinity for nucleic acids yielding less intense green fluorescence (Pierzynska-Mach et al., 2014 (link)). The vesicle red fluorescence intensity was quantified by using Fiji-ImageJ image processing software (Schindelin et al., 2012 (link)). At least 50 cells per each experimental condition were analysed, while corrected total cell fluorescence (CTCF), of both BSHE-untreated and BSHE-treated cells, was obtained as follows: CTCF = Integrated Density - (Area of selected cell x Mean fluorescence of background readings). (https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html#measuring-cell-fluorescence-using-imagej).

Adults were dissected in M9 medium. Eggs were collected and mounted on 2% agarose pads, covered by a coverslip and recorded at 23°C–unless otherwise stated. DIC movies were acquired using an AxioImager A2 Zeiss (objective lens 100×, NA 1.4) equipped with a Kappa camera and its accompanying time-lapse module. The resolution was 0.129 µm/pixel. Images were taken every 0,5 seconds and TIFF files were converted in Quicktime movies with the ImageJ software.

Cells were seeded in coverslips dishes at a density of 1 × 10⁴ cells/cm², maintained 24 h for attachment and then treated with BSHE extract (whose GR₅₀ concentrations were those referred to the 48 h-exposure).
After 48 h-incubation, cells were washed twice with PBS, fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.5% Triton X-100. Non-specific binding sites were blocked following a 30 min-exposure to 3% bovine serum albumin (BSA) in PBS-T (PBS and 0.05% Tween-20). Afterwards, cells were incubated with rabbit anti-LC3B antibody (dilution 1:200 in PBS and 1% BSA) at 4°C overnight. Cells were then washed three time with PBS-T and incubated with secondary Dy Light 488-conjugated anti-rabbit antibodies (dilution 1:1,000 in PBS and 1% BSA) in the dark at 4°C for 1 h and washed again with PBS-T.
Coverslips (overturned on microscope slides) were mounted with Pro Long Diamond Antifade containing DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride) (Thermo Fisher Scientific) and observed under fluorescence microscopy (Axio Imager A2 Zeiss). Images were acquired using a black and white camera (Leica, DFC350FX).

Rag1^–/–Rorc^gfp/+ mice were perfused with 20 ml 1 × PBS followed by 20 ml 4% paraformaldehyde (PFA) in PBS. Large intestines were washed three times with 1 × PBS at 4 °C, cut open, rolled up with inside out, and dehydrated in 30% sucrose overnight at 4 °C. Whole intestinal Swiss rolls were embedded in OCT (Tissue-Tek^®) at −80 °C for at least 6 h. The frozen tissue blocks were cut into 5 µm slices. After blocked with 1% BSA, sections were stained with anti-GFP-Alexa Fluor^®488 (Thermo Fisher Scientific) and 4,6 diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Sections were then observed on a fluorescence microscope Zeiss Axio Imager A2. Areas of cryptopatches were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Density of ILC3s in observed cryptopatch was calculated by the area of cryptopatches divided by the number of GFP+ cells.