Zen software package

Worms expressing GFP under control of tbb-6 (SLR115) and gst-4 (CL2166) were fed on NGM plates seeded with E.coli OP50. One- or two-day old adult worms were anesthetized in a drop of 2% sodium azide and images were captured immediately using a Zeiss AxioZoom v16 microscope equipped for fluorescence illumination. Fluorescence intensity was quantified using the Zeiss ZEN software package.

For indirect immunofluorescence, cells were grown on glass coverslips and fixed with ice-cold methanol or paraformaldehyde for 10 min, then permeabilized and immune-stained with appropriate antibodies. Fluorescence images were acquired using Nikon TE2000-U with Metamorph software (Molecular Devices). Immunofluorescence staining of PtdIns(4)P and PtdIns(4,5)P₂ was performed following published methods30 (link)31 (link)57 (link). The specificity of the PtdIns(4)P and PtdIns(4,5)P₂ antibodies was verified by pre-absorbing the antibody with PolyPIPosomes containing 5% of PtdIns(4)P or PtdIns(4,5)P₂ (Echelon) for 1 h at room temperature before incubation with samples.
Three dimensional structured illumination microscopy was performed following standard protocol. Briefly, an ELYRA Super-resolution Microscopy system (Zeiss) equipped with an alpha ‘Plan-Apochromat' 100 × /1.46 Oil DIC oil immersion objective and an Andor iXon 885 EMCCD camera was used to acquire raw images following standard protocols. Sections were acquired at 0.125-mm z-steps. Colour channels were aligned using alignment parameter from control measurements with 0.5-μm diameter multispectral fluorescent beads (Zeiss). Structured illumination reconstruction and image processing were performed with the ZEN software package (Zeiss). Final image processing was done using Adobe Photoshop (Adobe).

To stain the stereocilia of the saccule hair cells, embryos were initially fixed overnight in freshly thawed 4% paraformaldehyde at 4°C. Two washes for 5 min each in 0.2% PBSTx (1× PBS with 0.2% Triton X-100) were conducted at room temperature. Embryos were then incubated for 4 days in 2% PBSTx at 4°C to completely dissolve the otoliths. F-actin staining occurred for up to 2 h at room temperature when using Alexa-Fluor-488 tagged to phalloidin (Thermo Fisher Scientific, Eugene, OR, USA), which was diluted 1:20 in 1×PBS with Tween20 (PBSTw). Embryos were then washed six times for 10 min at room temperature in 0.2% PBSTx. Hair cell stereocilia were observed and imaged by using an LSM 780 inverted confocal microscope run and Zen software package (Zeiss).

For fluorescence and light microscopic analysis, 50 µl AMM on glass coverslips in a wet chamber was inoculated with 2 × 10⁴ conidia. Samples were analyzed after 0, 4, 6, 10, and 24 h using a Zeiss Axio Imager.M2 (Zeiss). Images were taken with an AxioCam MRm and analyzed by the use of AxioVision SE64 Rel. 4.9.1 imaging software (Zeiss). For confocal scanning laser microscopy, a Zeiss LSM 780 instrument was used along with an Airyscan detector where noted. Images were processed using the ZEN software package from Zeiss.
For scanning electron microscopy (SEM) analysis, resting conidia from mycelia grown on AMM agar plates for 5 days were collected using an electrically conductive and adhesive tag (Leit-Tab; Plano GmbH). Samples for resting conidia were fixed for 24 h in a desiccator containing a solution of 25% (vol/vol) glutaraldehyde, whereas swollen conidia were fixed in 2.5% (vol/vol) glutaraldehyde. Further preparation and scanning electron microscopy were carried out as previously described (56 (link)).