Permanox slide

Myricetin (Myr), myricitrin (Myr-gly), quercetin, vancomycin hydrochloride and purified α-Hla were purchased from Sigma-Aldrich (USA). For in vitro assays, stock solutions were prepared in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, USA) while for in vivo studies, the substances were dissolved in sterile phosphate-buffered salt (PBS) buffer pH 7.0. Sterile 96-well polystyrene flat-bottom microtiter plates (Costar 3599) were purchased from Corning Inc. (USA) and hydrophobic modified polystyrene (Permanox™) slides were purchased from NalgeNunc International (USA). A 10 µL Hamilton® Microliter™ syringe was used to inject inoculum aliquots into G. mellonella.

The 5 × 10⁴ OKG4 cells/well in DermaLife medium containing 60 μM Ca²⁺ were seeded to 8-well Permanox slides (Nunc) and cultured for 24h. The cells were subsequently cultivated in 60 μM or 1.4 mM Ca²⁺ for further 24 h and treated with the respective cell culture medium containing ICC-coupled CMS 10-E-15-350 at a final ICC concentration of 2 μg/ml for further 24 h. The cell nuclei were stained with Hoechst 33342 dye (blue, Invitrogen) and cell membranes were stained with Wheat Germ Agglutinin (WGA) Alexa Fluor 488-conjugated (Invitrogen), which binds to N-acetylglucosamine and N-acetylneuraminic acid moieties on the cell surface, for 30 min. Subsequently, the cells were rinsed with PBS to remove the excess of dyes. The internalization was observed using the LSM700MAT (Zeiss) and analyzed by the ZEN software (Zeiss). Each analysis was performed in triplicate.

DRGs were plated in spot cultures in 8-well Permanox slides (Lab-Tek, Thermo Scientific). Neurons were fixed in 4% paraformaldehyde, washed, and blocked as described (Shin et al., 2012 (link)). NMNAT2-myc was detected with anti-myc tag antibody (9B11; CST #2276), and neurons were co-stained with anti-β3 tubulin. Larval filet preps of third instar larva were fixed in 4% paraformaldehyde for 20 min at room temperature. Blocking and staining were performed in PBS + 0.1% Triton X-100 + 5% goat serum. All Drosophila samples were mounted and imaged in 70% glycerol containing Vectashield. Samples were imaged on a Leica DMI 4000B Confocal microscope using 40x or 60x oil objectives. Images are maximal Z-projections of confocal stacks. Samples for each experiment were processed simultaneously with identical confocal gain settings. Intensity of NMNAT2-myc and SCG10 in DRGs was quantified using ImageJ by determining the mean grey value within the total axonal area as defined by Tuj1 staining with background subtracted. A similar strategy was employed for intensity of HA-dNMNAT, with HRP labeling total axonal area.

Human bladder grade II carcinoma cells (5637, ATCC# HTB-9) were cultured, to 70–80% confluency on 8-well chamber Permanox slides (6x10⁴ cells/well), in 6-well plates (6x10⁵ cells/well) or 96-well plates (5x10⁴ cells/well), (all from Thermo Scientific) in RPMI-1640 supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids and 10% heat-inactivated FBS (PAA) at 37°C with 5% CO₂. Gentamicin (50 μg/ml) was from GE Healthcare.

Spore solutions of the WT, Δgpi7, and Regpi7 strains were prepared in 0.1% Tween 20 in saline at a concentration of 1 × 10⁸ conidia/ml. Ten microliters of each spore solution was added into 200 μl of CM medium sitting on a microscopy slide and mixed well by pipetting and stirring. Two different hydrophobic surfaces were used: polystyrene cell culture slides and Permanox slides (Thermo Scientific Nunc). Ordinary glass microscopy slides were used as a non-hydrophobic surface. The slides were incubated at 37°C for 0–6 h, the culture medium was removed, the slides were washed shortly in 0.1% Tween 20 in saline, and the adherent spores were removed by running 1 ml 1% Tween 20 in saline over the slide. For each slide, this solution was collected in an E-cup, appropriate dilutions in 0.1% Tween 20 in saline were prepared, and 100 μl of each dilution was spread on the CM plate and incubated at room temperature for 2 days, after which the number of colony-forming units was determined. For statistical significance, each experiment was performed five times for each strain and condition.