Clsm 800

Mock- or RSV-infected A549 cells were stained with MitoTrackerRed CMXRos (M7512, ThermoFisher Scientific; 100 nM, 15 min) and then fixed, washed and stained using standard protocols (Hu et al., 2013 (link)). Primary antibodies used were: anti-RSV antibody (1:400, ab20745, abcam), anti-γ-tubulin antibody (1:300, 66320–1-Ig, Proteintech), or anti-α-tubulin (1:100, sc-5286, Santa Cruz), with dye-tagged secondary antibodies (anti-goat Alexa Fluor 488, 1:1000, A-11055, or anti-mouse Alexa Fluor 647, 1:1000, A-31571, ThermoFisher Scientific) as appropriate. F-actin was stained by Alexa Fluor 488 phalloidin (1:1000, A12379, ThermoFisher Scientific). In all analyses of stained fixed cells, nuclei were stained by DAPI (1:15,000 in PBS, 10236276001, Sigma). Following mounting onto glass slides with Biomeda Gel Mount (ProSciTech), imaging was conducted using a Leica TCS SP5 channel confocal and multiphoton microscope (63X objective, oil immersion). Images (512 × 512 pixels, 8- or 12-bit) were collected and viewed using the Leica Application Suite Advanced Fluorescence Lite Version: 2.8.0 build 7266 viewer software. Airyscan super-resolution imaging was performed using the Zeiss CLSM 800 with Airyscan detector; images (2448 × 2448 pixels, 16-bit) were viewed using the ZEN 2 (blue edition) software.

N. benthamiana plants were grown in 8 cm³ pots filled with soil S-Jord (Hasselfors) under controlled growth conditions of 16 h light 8 h dark cycles, 70% relative humidity, light intensity of 150 μE m^–2 s^–1, and day and night temperature of 22 °C and 20 °C, respectively. Transformed A. tumefaciens strain GV3101 (Rifampicin, 100 μg/mL) carrying the constructs of interest were grown in 5 mL of Luria-Broth high salt medium (L1704, Duchefa Biochemi, Haarlem, The Netherlands), supplemented with appropriate antibiotics (see Table S4, destination clones for expressing fluorescent fusions in plants). Liquid bacterial cultures were shaken at 200 rpm, 28 °C overnight, and then sedimented at 4000 G for five minutes. The resulting pellets were resuspended in MQ water with 150 μM Acetosyringone to the final OD₆₀₀ = 0.15 and infiltrated in the abaxial side of leaves of five-week-old N. benthamiana plants. The leaves were imaged on the third day post-infiltration using CLSM800 (Carl Zeiss), objective C-Apochromat 40×/1.2 W, excitation light 488 nm and 561 nm and emission ranges of (515–560 nm) and (570–650 nm) for GFP and RFP, respectively. Images were analyzed using ZEN black software (Carl Zeiss).

Images were taken with a Zeiss laser scanning confocal microscope (CLSM800) equipped with a Zeiss 63× (1.4 NA) plan-apochromatic oil differential interference contrast objective in sequential mode with a resolution of 1024 × 1024 pixels. The laser intensity and the detector gain were adjusted to values that avoid signal saturation, and all images of one experiment were imaged with the same settings in one continuous session. The images were quantitatively analyzed using the ImageJ software (version 2.3.0).
For quantification of cell surface staining, the outer and inner perimeter of the cell surface were exactly outlined. Then, the fluorescence intensity value obtained from the inner border was subtracted from the one of the outer border so that only the fluorescence present at the cell surface was determined. For quantification of the total cell staining, the outer border of the cell was marked, and the mean fluorescence intensity was measured.
For quantification of in situ PLA signals, the soma of neurons or HEK-293 cells was surrounded, and the fluorescent dots inside these borders were counted using the ImageJ option “Find maxima”. A fixed noise tolerance value was used for the analysis of all images of the same experiment. The PLA signals were normalized to the area analyzed and to the GABA_B receptor expression level.