Dmrxa fluorescence microscope

Each individual grouper, E. coioides, was starved for 24 h and then intraperitoneally injected with 40 μl of 10⁷ TCID₅₀/ml RGNNV. Grouper fry injected with PBS served as the controls. Each group contained 30 grouper fry. After 1 week, the brain tissues of each group were collected and fixed in 10% neutral-buffered formalin. The fixed tissue sections were stored before the subsequent experiment. The frozen tissue sections were incubated with TAMARA labeled aptamers (200 nM) at 4°C for 1 h in a darkroom. After the samples were washed with PBS, the tissues were imaged with a Leica DMRXA fluorescence microscope. Tissue sections incubated with the TAMARA-labeled initial library were used as the controls.

Culture samples were obtained and processed to analyse cell viability and sporulation at various incubation time points, as previously described [43 (link)]. Cell viability and sporulation were analysed by staining the cells with propidium iodide and SYTO 9 (LIVE/DEAD Bac-Light Bacterial Viability Kit, Invitrogen, L-13152, Waltham, MA, USA). The samples were observed under a Leica TCS-SP8 confocal laser-scanning microscope with wavelengths of 488 and 568 nm excitations and 530 nm (green) or 640 nm (red) emissions [43 (link)] for SYTO 9/PI staining.
SCO2102-mCherry and SCO2103-eGFP were analysed in SFM plates without cellophane. After spore inoculation, cover glasses were positioned at an angle of 45 degrees. At the indicated time-points, the cover glasses were removed and the samples were mounted with ultrapure mQ water. Then, they were observed using a Leica DMRXA fluorescence microscope with FITC and Texas Red filters. Pictures were taken with an ORCA-Flash4.0 V3 Digital CMOS camera.
Culture controls without SYTO 9/PI staining and without eGFP or mCherry were used to fix the levels at which autofluorescence was detected.
Microscopy images were processed (histogram intensity levels were adjusted, and the scale was added) using Fiji software [44 (link)]. Figure composites were made using AdobePhotoshop CS5.1.

HeLa cells were propagated on culture microscope slides under the conditions described above. After incubation for 24 h, the cultures were washed three times with phosphate buffered saline (PBS), fixed with acetone for 20 min at −20°C, washed with the same buffer and incubated overnight at 4°C with appropriate dilutions of the primary antibodies (Table 1). The slides were then washed for 30 min with PBS, placed in the dark and incubated with the secondary antibodies (Table 1) for 90 min in a humid chamber. The samples were washed three times with PBS and incubated successively with 1 μg/ml phalloidin-TRITC conjugate (Sigma-Aldrich) for 90 min and 10 ng/ml DAPI (Southern Biotech; Birmingham, USA). The preparations were visually examined and photographed in a Leica DMR-XA fluorescence microscope coupled to Leica Qfluoro software in the Image Processing facility of the University of Oviedo. The quantification of fluorescence for the subsequent statistical analysis was carried out using ImageJ analysis software (28 ).