Application suite advanced fluorescence

The fluorescence images of transgenic tobacco expressing nat-T-phyllo-GFP and control tobacco plants were captured with confocal laser scanning microscope (TCS SP5; Leica Microsystems CMS GmbH, D-68165 Mannheim, Germany) using LAS AF (Leica Application Suite Advanced Fluorescence) 1.8.1 build 1390 software. We used the PL FLUOTAR objective (10.0X/N.A.0.3 DRY) with confocal pinhole set at Airy 1 and 1x zoom factor for improved resolution with eight bits. GFP expressed in transgenic plants was excited with an argon laser (30%) with AOTF for 488 nm (at 40%) [32 (link)], and the fluorescence emissions were collected between 501 and 580 nm with the photomultiplier tube (PMT) detector gain set at 1150V.

Following cisplatin damage, cells were either processed immediately, or medium was replaced and DNA repair allowed for indicated periods. Cell extraction was carried out in situ by washes of 0.1% Nonidet P-40 for 10 minutes on ice to remove all soluble proteins. Following fixation in 4% paraformaldehyde and cell permeabilization with 0.3% Triton X-100, cells were blocked overnight in 10% donkey serum at 4 °C. After incubation with indicated primary and secondary antibodies, cells were mounted with Prolong Gold antifade. Proximity ligation assay (DuoLink, Sigma) was performed using the manufacturer’s instructions. All fluorescence images were obtained using a Leica DMI 6000 confocal microscope using ×100 objective (1.4 numerical aperture) with LAS AF 2.7.2.9586 software (Leica Application Suite Advanced Fluorescence). Maximum intensity images from focal plane z-stacks (spaced 0.2 µm apart) were acquired and deconvoluted. Quantification of fluorescent signal were performed using Image J software.

Cells (4 × 10⁴) were plated on eight-chamber plates (Ibidi) and incubated with RPMI 1640 containing 10% FBS for a day. The medium was replaced with phenol red- and serum-free RPMI 1640 containing 10 μM HMRef-βGal. The cells were incubated for 1 h, and differential interference contrast (DIC) and fluorescence images were obtained using a Leica Application Suite Advanced Fluorescence with a TCS SP5 X and a dry objective (× 10, numerical aperture 0.40; Leica). The light source was a white-light laser.

The cells were cultured in a 12-well plate with cover slides according to the density of each well of 2 × 10⁵. Twenty-four hours later, the cells were infected with SARS-CoV-2 (MOI = 0.5) or transfected with 3 μg of plasmid. Cell samples were collected according to the experimental design. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 min, blocked with 5% BSA, and permeabilized with 0.1% Triton for 45 min. The treated cells were incubated with the primary antibody at room temperature for 2 h and then with the secondary antibody was incubated for 1 h. Glass coverslips on the slides were sealed by dripping the DAPI stain and sealing agent. Cell samples were analyzed by a Leica SPR8 laser confocal microscope. For FLIM/FRET experiments, the “donor only” sample was transfected with G3BP2 plasmids. We analyzed the colocation between G3BP2 and TRIM25 with N protein exist or not. The average fluorescence lifetimes of the donor-only and FRET samples were measured in fast FLIM mode. The measurement was completely controlled via Leica Application Suite Advanced Fluorescence. The FRET efficiency was calculated from the ratio of the FRET donor lifetime τ_quench and the non-FRET lifetime τ_quench as follows:

Four sections adjacent to identified graft sites and co-labeled for SC121 and ChAT+ host motor neurons were imaged at 10x using the Leica DFC365 FX camera, Leica DM6000 B microscope, and Leica Application Suite Advanced Fluorescence 3.2.0.9652 program. Images from treated animals (n = 8, average of at least eight sections per animal) and untreated animals (n = 6, average of four sections per animal) were used for motor neuron counts using the Freehand Selections tool and Region of Interest Manager in ImageJ. Images also underwent automated size analysis of motor neuron areas using IMARIS software with manual thresholding. Untreated (n = 2) and treated (n = 2) animals were removed from the IMARIS analysis due to insufficient contrast in the ChAT staining for the software to accurately distinguish positive staining from background.