Dmrxa

We adapted a standard upright widefield fluorescent microscope (Leica DM RXA) for SMLM acquisition. A 405 nm laser (LuxX + 405–120, Omicron) and a 488 nm laser (LuxX + 488–100, Omicron) were used for activation and excitation, respectively, which were coupled in using a polarising-maintaining optical fibre, all from Omicron-Laserage Laserprodukte GmbH, Germany. Standard Leica fluorescent cubes were used (FITC filter or N2.1 filter), with the excitation filters removed from the filter blocks. Fluorescence was collected using either a long-working distance lens (HCX PL Fluotar L 100x/0.75, Leica) in combination with a cryostage (CMS-196, Linkam Scientific, UK), or an oil-immersion lens for experiments at 294 K (PL Fluotar 100x/1.3, Leica). Fluorescent images were recorded by a sCMOS camera (pco.edge 4.2, DVision, Oostakker, Belgium).
Fluorescence emission spectra were recorded by a home-made set-up that was mounted on an extra camera port via a beam-splitter (100-50-0%, Leica). The emitted light was coupled in a multimode optical fibre (M15L01, Thorlabs Inc, USA) by an aspheric glass lens (C340 TMD-A, Thorlabs Inc, USA) and connected to a fibre-optic spectrometer (AvaSpec-2048L, Avantes, the Netherlands). Software written in-house was utilised to read-out the camera, and to synchronise the lasers and camera acquisition.

Live and fixed yeast cells were captured using Leica DMRXA with the Metamorph software or with a Deltavision microscope with deconvolution. Aggregate containing cells were scored with Metamorph or ImageJ Cell Counter plugin (at least n = 50 cells per condition). Nuclei were stained with 200 ng/ml DAPI (Sigma). HeLa cells were cultivated on coverslips and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 in PBS and stained after blocking with 1% BSA in PBS. Primary anti-tubulin antibodies (dilution 1:100), were incubated onto the cells for 2 h at room temperature. After washing, the secondary antibodies directed against mouse IgG were incubated for 1h at room temperature. The nuclei were stained with DAPI/Antifade (Q-BIOgene MP Biomedicals, Illkirch, France). Samples were examined using a Deltavision microscope.

Cells grown on glass coverslips were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 in two consecutive steps, each at room temperature for 15 min. After washing with PBS, cells were incubated in 10% FBS (diluted in PBS) for 30 min to block unspecific signals. After this step, cells were incubated with diluted primary antibodies at room temperature for 1 h and then extensively washed with PBS/0.1% Tween 20. The incubation with secondary antibodies was performed at room temperature for 1 h. To counterstain nuclei, coverslips were mounted in Mowiol containing 4′,6-diamidino-2-phenylindole (Sigma) and viewed by a fluorescence microscope (Leica DMRXA).

Gills were excised from grayling and rainbow trout and fixed in a 4% paraformaldehyde solution in phosphate-buffered saline overnight at 4°C. Gills were washed in phosphate-buffered saline and underwent ethanol dehydration prior to paraffin wax embedding. Microtome sections of gill tissue (7 μM thick) were fixed onto Superfrost^® plus microscope slides (Thermo Fischer Scientific, Rockford, IL, USA) and were stained with haematoxylin and eosin and examined under light microscopy (Leica DM RXA). Digital images were collected from three randomly selected fish, and two filaments from each fish with 10 adjacent lamellae were imaged (QI Click, QImaging). ImageJ software (National Institutes of Health) was used to calculate the interlamellar cell mass (ILCM) to lamellar length ratio as previously described (Ong et al., 2007 (link)). The height of the ILCM was measured parallel to the total lamellar length, starting from the edge of the ILCM bordering the filament to the most distal edge of the ILCM from the filament. Ratios were calculated and compared among the three treatments for both grayling and trout and analysed via one-way ANOVA (Prism 6; Graphpad Systems).

The specificity of aptamer-STIV binding was further verified by fluorescent localization, as described previously [30 (link)], with some modifications. STIV was labeled with the aptamers as follows: FITC-aptamers were denatured at 95 °C for 5 min and cooled on ice for 10 min, followed by the addition of 100 μl purified STIV (10⁹ TCID₅₀/ml) and incubation on ice for 1 h. The aptamer-STIV mixtures were passed through a pre-wetted 0.1-μm PVDF filter (EMD Millipore) and eluted from the filter by washing with TN buffer. The chemical dye Hoechst 33,342 was then added to label the virus. After washing three times, the samples (10 μl) were dropped onto a glass coverslip and imaged using a fluorescence microscope (Leica DMRXA, German) at an excitation wavelength of 488 nm (green for FITC) or 350 nm (blue for Hoechst 33,342). The FITC-library pool and SGIV served as controls.