Emitech k850

Fixed samples of anthers with pollen grains were dehydrated in a successive 15, 30, 50, 70, 90, and 99.5% acetone series for 15 min at room temperature and twice in anhydrous acetone. Next, the pollen grains were critical point dried in liquid CO₂ using an Emitech K850 dryer (Quorum Technologies Ltd., Ashford, United Kingdom). Dried grains were transferred onto the microscope stage and sputter-coated with gold using an Emitech K550X device (Quorum Technologies Ltd., Ashford, United Kingdom). Observations of the surface of pollen grain sculpture (striae, perforations, microstriae) and photographic documentation were made using a Tescan Vega II LMU scanning electron microscope (SEM) (Tescan Orsay Holding, Brno, Czech Republic). The microscope was used for analysis of 100–150 pollen grains from each cultivar.

Fixation of treated cells was performed using 2.5 % glutaraldehyde. Thereafter, cells were dehydrated in a graded ethanol series (70, 90, 100, 100, 100 %, and acetone), desiccated by critical point drying (Emitech K850; Quorum Technologies LTD, West Sussex, UK), mounted, sputtered with gold–palladium (Polaron CA 508; Fisons Instruments, Mainz-Kastel, Germany) and examined with a JEOL JSM—5400 scanning electron microscope (Eching, Germany).

For scanning electron microscopy (SEM), fresh cultures of G. duodenalis trophozoites WB6 strain were treated with either rBSH47 (0.5 μg/ml), rBSH56 (0.08 μg/ml), or DCA (0.1 g/L and 0.2 g/L) in KM supplemented with 10% heat-inactivated FCS (10%), with or without bovine bile (0.6 g/L) supplementation. Cultures of treated and untreated Giardia trophozoites were subsequently seeded in 12 wells plates on poly-lysine glass coverslips placed at the bottom, and parasites were let to settle on the glass coverslips. After 16 h incubation, the supernatants were removed gently and cells were fixed on the glass coverslips with cacodylate 0.1 M and glutaraldehyde 2.5% (pH 7.2) overnight at 4°C. After two washing steps with 0.1 M cacodylate (pH 7.2), cells were dehydrated in a graded ethanol series (50, 70, 90, and 100%) and critical point-dried in liquid CO₂ (Emitech K850, Quorum Technologies). Coverslips were then mounted onto holders and coated with 20 nm of gold (JEOL Fine Coater JFC-1200). The samples were then examined with a Hitachi Scanning Electron SU3500 Premium.

Samples for CLSM imaging and analysis were fixed with phosphate-buffered formaldehyde solution (4%, Roti®-Histofix, Carl Roth GmbH, Karlsruhe, Germany) for 12 h at 4°C and afterwards air-dried at room temperature in the dark. During preparation, samples were handled with care to prevent detachment and loss of adhered cells and biofilm, e.g. due to shear forces during rinsing. Dried samples were mounted on microscope slides, embedded using the ProLong® Antifade Kit (P7481, Molecular Probes®, Darmstadt, Germany) according to the manufacturer’s instructions and sealed with cover slips to prevent photo-bleaching effects during fluorescence microscopy and to enable long-time storage of the samples at −20°C. For SEM imaging, biofilm samples were fixed with glutardialdehyde solution (2.5% in PBS, pH 7.0) at room temperature for 12 h. After fixation, samples were carefully washed for 10 min in PBS buffer solution and twice in distilled water. Subsequently, samples were dehydrated using an ascending ethanol series from 10 to >99.9% with 10 min incubation for each step and two times 30 min for the last step. The dehydrated bacterial samples were critical-point dried (EMITECH K850, Quorum Technologies Ltd, East Grinstead, UK) and gold sputter coated (∼ 5 nm) (S150B, Edwards Ltd, Crawley, UK).

Isolated parasites were first observed on slides using light microscopy. Images were acquired using a Nikon DXM 1200C camera and a micrometric slide to set the scales, and the images were processed using ImageJ software (https://imagej.nih.gov/ij/). In parallel, pools of isolated and washed gamonts and gametocysts and relevant sections of infected acridian ceca and midguts were prepared for scanning electron microscopy (SEM). After appropriate washing in 0.22 μm-filtered sterile PBS 1X, the samples were fixed in 5% (v/v) glutaraldehyde in 0.2M cacodylate buffer (pH 7.2) at 4 °C for 6–12 h then washed twice in 0.2M cacodylate buffer (pH 7.2) before undergoing successive series of dehydration in 50, 70, 90 and 100% ethanol. Samples were critical point-dried in liquid CO₂ (Emitech K850, Quorum Technologies, Lewes, United Kingdom) then coated with 20 nm gold (JFC-1200 Fine coater, JEOL, Tokyo, Japan). Samples were then examined with a Hitachi Scanning Electron Microscope SU3500 Premium (Hitachi, Tokyo, Japan), as previously described [2 (link)]. Quantitative measurements were length and width at the different life stages, including length of protomerites and deutomerites for trophozoites and gamonts.

For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 m phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 (Quorum Technologies, East Sussex, UK), coated with platinum using a Quorom Q150R S sputter coater (Quorum Technologies). and visualised using a JEOL LSM-6010 scanning electron microscope (Jeol, Herts, UK).
Transmission electron microscopy was performed as previously described.^{22 (link)}

CPC adhesion on scaffolds was analyzed by SEM at 7 and 14 days, following a reported procedure [43 (link), 44 (link)]. Briefly, constructs were fixed in 3% glutaraldehyde solution (Sigma-Aldrich, Italy) for 15 min at room temperature, followed by post-fixation with 1% osmium tetroxide (Sigma-Aldrich, Italy) for 15 min. After washing in PBS, specimens were dehydrated by graded alcohol series, followed by critical point drying (Emitech K850, Quorum Technologies, UK). Specimens were mounted on aluminium stubs with adhesive carbon tape and gold-sputtered prior to observation performed with an acceleration voltage of 5 kV at a working distance of 13 mm.