Cpd300

The structure and morphology of cells on the surface of the models are important to define the health status of the models. Scanning electron microscopy (SEM) was used to visualise the morphology of cells on the apical side of membrane inserts. Briefly, the models were washed twice with PBS and fixed with 2.5% glutaraldehyde in PBS (cat: G5882, Sigma Aldrich) for 2 h. Models were washed with PBS for three times, each time for 5 min. Next, the membranes of the models were cut from the insert, dehydrated in graded ethanol solutions (from 30 to 100% ethanol) and dried using a critical point dryer (CPD300, Leica Microsystems). Models were then coated with gold at 10 nm coating thickness. Subsequently, the samples were kept in a desiccator filled up with silica gel prior to SEM imaging. SEM imaging was performed using Zeiss Sigma HD FEG SEM. Secondary electrons were acquired using the detectors of the SE2 mode for all models with an accelerating voltage of 5.0 kV and a working distance (W.D) = 8.0.

C. auris [DSM 21092] was incubated (37 °C, 24 h) with Rub C at the concentration of 250 and 15.6 μg/mL in 96-well non-tissue microtiter plates in RPMI 1640 medium supplemented with 0.165 mM MOPS. Cultures were fixed with 5% formaldehyde and 2% glutaraldehyde (final concentrations) and washed twice in TE buffer (20 mM TRIS with 1 mM EDTA, pH 6.9). A 50 µL aliquot was added to round poly-l-lysine pretreated coverslips and incubated (room temperature, 10 min). Further processing was carried out as previously described with slight modifications [45 (link)]. In brief, cells were fixed for 10 min on the coverslip with TE buffer including 1% glutaraldehyde (final concentration). The coverslips were washed twice in TE buffer and dehydrated in 10 min steps on ice with a graded series of acetone (10%, 30%, 50%, 70%, and 90%), followed by two steps in 100% acetone at room temperature. The coverslips were mounted onto aluminum stubs with carbon adhesive discs; they were critical-point-dried with the automated CPD300 (Leica Microsystems) and gold-palladium-sputter-coated (55 s at 45 mA) with a SCD500 (Bal-Tec, Balzers, Liechtenstein). Images were acquired with a field emission scanning electron microscope Zeiss Merlin (Zeiss, Oberkochen, Germany) using the Everhart Thornley HESE2 detector and the in lens SE detector in a 25:75 ratio with an acceleration voltage of 5 kV.

Leaves of TH-0%, TH-75%, and CK groups were cut into pieces of about 3 mm × 3 mm size with a blade, quickly placed into 2.5% glutaraldehyde solution, fixed for 2 h at room temperature after pumping air to make the tissue sink, rinsed using the phosphoric acid buffer for three times, subjected to gradient dehydration with ethanol (50, 70, 80, 90, 95, and 100%), dehydrated with anhydrous ethanol, and dried using critical point dryer (CPD300, Leica Microsystems, Wetzlar, Germany). Then, the sample was pasted on the cover glass, put into the vacuum spray machine for spraying, and finally observed and photographed by scanning electron microscope (HITACHI SU3900, Shimadzu Corporation, Tokyo, Japan). The number of stomata was observed under the scanning electron microscope, and three replicate samples were used for each treatment group. The stomatal density (pieces/mm² = number of stomata in the field of view/field area) was calculated by measuring the field of view using a micrometer scale and calculating the field of view.