Lsm 880 meta confocal microscope

Retinae and primary RGCs immunofluorescence analysis was performed as previously described [6 (link), 33 (link)]. In brief, retinal sections or cells were incubated with primary antibodies (1:100 diluted in 1% Normal donkey serum in PBS) at 4 °C overnight, followed by incubating with secondary antibodies (1:100 in PBS) at 37 °C for 1 h. Nuclei were counterstained with DAPI. Images were obtained by a Carl Zeiss LSM880 Meta confocal microscope. Statistical quantitative analyses of retinal immunostaining were performed as previously described [33 (link)]. Briefly, the intensity of immunofluorescence staining was quantified by measuring the area of ganglion cell layer (GCL) and inner plexiform layer (IPL) under the curvilineal diagrams plotted with Origin 8.0 software. Data from three independent curvilineal diagrams were averaged for each eye.

For the analysis of the antiviral activity of compounds, cells were preincubated with compounds, infected, and incubated for 48 h prior to immunofluorescence staining. For the cell-cell fusion assay, 293T/hACE2 cells were incubated with 293T cells transfected with the SARS-CoV-2 spike protein in the presence or absence of ATRA. The cocultured cells were incubated for 24 h, and the state of cell fusion was observed by microscopy and immunofluorescence staining.
Cells were washed three times in PBS and fixed with 4% paraformaldehyde (PFA) for 2 h at room temperature. Then, the cells were permeabilized with 0.1% Triton X-100 for 30 min, washed with PBS, blocked with 2% bovine serum albumin (BSA) for 1 h, and stained with anti-SARS-CoV-2 nucleocapsid (Abcam catalog no. ab271180), anti-SARS-CoV-2 spike (Abcam catalog no. ab273433), and anti-hACE2 (Abcam catalog no. ab108252) antibodies for 2 h at room temperature, according to different experimental settings. The cells were washed with PBS and then stained with secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (Abcam catalog no. ab228549). Images were examined by a Zeiss LSM 880 Meta confocal microscope.

Full-length MdSYP121 and MdSNAP33 were fused to yellow fluorescent protein (YFP) vectors to pUC-SPYCE-35S and pUC-SPYNE-35S, respectively, by T4 recombination. The primers used are listed in Supplementary Table S1. These two recombinant plasmids were transiently expressed in tobacco leaves by A. tumefaciens (GV3101)-mediated infiltration^{28 (link)}. The YFP fluorescence of tobacco leaves was detected after infiltration for 4 days using a LSM 880 META confocal microscope (Carl Zeiss, Germany).

The open reading frames (ORFs) of MdSYP121 and MdSNAP33 were inserted into a pCB302 green fluorescent protein (GFP) vector, whose N-terminus consists of a GFP under the control of the CaMV 35S promoter. To achieve transient expression, the recombinant plasmids pCB302-MdSYP121-GFP and pCB302-MdSNAP33-GFP were transformed into A. tumefaciens GV3101. After the cell cultures were incubated overnight, A. tumefaciens cells were harvested via centrifugation and resuspended in infiltration medium (100 mL of medium containing 1 mL of 1 M MES-KOH at pH 5.6, 333 μL of 3 M MgCl₂ and 100 μL of 150 mM acetosyringone). The leaves of 5-week-old N. benthamiana plants were used for transient expression, and the GFP signals were observed using a LSM 880 META confocal microscope (Carl Zeiss, Germany). A pCB302-GFP construct was used as a control.

Differentiated NSCs (DIV7) were incubated with MitoTracker Red (Thermo Fisher Scientific; M7512) at a final concentration of 10 nmol/L for 15 minutes before live cell imaging analysis. Live cell imaging was performed using a Zeiss LSM 880 Meta confocal microscope with an enclosed climate chamber. Cells were maintained at 37°C in 5% CO₂ /95% air during imaging. To generate kymographs, time‐lapse images were recorded every second for 2 minutes. Quantitative analysis of mitochondria velocity was performed using IMARIS software (BITPLANE). Axons were designated as regions of interest (ROI) in IMARIS. Particles were identified as mitochondria, and all of the mitochondria in a given ROI were tracked. To select the velocity ranges which correctly represented the mitochondrial movement in differentiated NSCs, instantaneous velocities of each mitochondrion at each time point were plotted as a histogram with a 0.5 mm/s interval. ImageJ was used to produce Figure 3A.