Polysine adhesion slide

Fresh, unfixed C57BL/6 mouse brains (N = 3) were embedded in tissue freezing medium (Leica Biosystems, Richmond, UK), frozen on a dry ice ethanol bath, sectioned at 20 μm on a cryostat, and then mounted onto Polysine Adhesion Slides (Thermo Fisher Scientific, Waltham, USA). RNA probe hybridization and subsequent washes were performed as described previously^{56 (link)}. Fluorescein-labeled probes were detected using peroxidase-conjugated anti-fluorescein antibodies (Roche Diagnostics, Mannheim, Germany). Peroxidase activity was detected using Cy3-tyramide conjugate. After the detection, peroxidase activity was blocked by 100 mM glycine-HCl (pH 2.0) solution containing 0.1% Tween. Sections were washed with 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl and 0.05% Tween and then blocked by 10% goat serum and 1% blocking reagent powder (Roche Diagnostics, Mannheim, Germany) in 0.1 M Tris-HCl (pH 7.5) with 0.15 M NaCl. Dioxygenin (DIG)-labeled probes were detected using peroxidase-conjugated anti-DIG antibodies (Roche Diagnostics, Mannheim, Germany). Peroxidase activity was detected using fluorescein-tyramide conjugate. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Munich, Germany).

Hemolymphs of 50 infected D. citri insects were collected and then were placed on Polysine adhesion slides (Thermo Fisher Scientific) and dried at room temperature. The samples were fixed in 4% paraformaldehyde for 2 h and treated with 0.2% Triton X-100 for 1 h as previously described (12 (link)) and then were immunolabeled with P8-FITC or actin dye phalloidin-Alexa Fluor 647 carboxylic acid (Thermo Fisher Scientific). The treated samples were examined with a Leica TCS SPE inverted confocal microscope.
Organs of D. citri insects were successively dissected, fixed, and treated with Triton X-100, as mentioned above. The samples then were immunolabeled with P10-FITC and finally detected using immunofluorescence microscopy.

Anaesthetized rats were euthanized by intracardial injection of lethabarb (Virbac Pty.Ltd., NSW, Australia) immediately prior to tissue collection. For orientation, the dorsal region of the eye was marked with a tattoo pen, eyes were removed, the anterior components dissected, and the posterior eye cup placed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 30 min. The eye cups were subsequently cryoprotected in graded sucrose solutions (10, 20, 30% v/w) and snap frozen and stored at −80°C until use.
Frozen sections (14 μm) were cut on a cryostat (Hyrax C60, Zeiss, Germany), mounted onto poly-L-lysine coated glass slides (Polysine® Adhesion slides, Thermo Scientific, VIC, Australia) and stored at −80°C until use. All sections were cut from the dorsal to ventral region of the eye, as close to the optic disc as possible, to allow for consistency between eyes.

Cells for immunofluorescence were cultured on flamed glass coverslips coated with 7.5μg/ml fibronectin (for adherent culture), or cytospun onto polysine adhesion slides (Thermo Fisher) using a Shandon Cytospin 3 centrifuge (for sorted samples in suspension). They were fixed in 4% formaldehyde/PBS, quenched with 50mM ammonium chloride, blocked in 3% donkey serum and 0.1% Triton X-100. Cells were then incubated with primary antibody for 3 h at room temperature, washed 3 times with PBS, incubated with secondary antibody and/or 100ng/ml DAPI for 1h at room temperature, washed 3 times with PBS, mounted in ProLong Gold Antifade Mountant (Molecular Probes), and imaged on a Leica SP8 confocal microscope. Where recommended by antibody manufacturers, a methanol permeabilisation step was included prior to blocking.

The potential role of ISDPs in membrane permeabilization in living C. albicans SC5314 cells was studied by fluorescence microscopy using a Nikon Eclipse 80i optical microscope, equipped with a Nikon Digital Sight DS-2Mv camera, and images were acquired with NIS Elements F control software (Nikon Co., Tokyo, Japan). Yeast cells grown in yeast extract, peptone, and dextrose broth overnight at 30 °C with shaking (100 rpm) were washed once with water and 4 × 10⁷ cells/mL were loaded with 500 μM Lucifer Yellow (LY) and 1.5 μM propidium iodide (PI). LY is a fluorescent molecule used as a quantitative marker of the cell membrane permeabilization [23 (link)], while PI is a non-vital nuclear stain commonly used for identifying dead cells. Yeast cell suspensions (10 μL) were seeded on Polysine Adhesion Slides (Thermo Scientific™, Thermo Fisher Scientific Inc., Waltham, MA, USA) and, after 10 min, ISDPs were added (final concentrations in the range 90–97 μM). Images were taken up to 30 min at a 40× magnification.

After separation, the cells collected from all outlets were labeled using immunofluorescence staining to identify tumor cells and WBCs. Specifically, cells were first obtained by the centrifugation of collected samples and coated onto Polysine adhesion slides (P4981, Thermo Fisher Scientific) at room temperature. The adherent cells were then fixed with a −20 °C methanol solution for 5 min. After incubation in PBS buffer with 10% normal goat serum (Abcam), the tumor cells were stained with fluorescein isothiocyanate (FITC)-conjugated Pan-CK monoclonal antibody (Thermo Fisher Scientific), while the WBCs were stained with allophycocyanin (APC)-conjugated CD45 antibody (BioLegend) at 4 °C overnight. The fluorescently labeled cells were mounted using Prolong Gold Antifade Mountant with DAPI (Sigma‒Aldrich). Cells that were positive for Pan-CK and DAPI but negative for CD45 were identified as tumor cells, while cells that were positive for CD45 and DAPI but negative for Pan-CK were identified as WBCs.