Tcs sp8 confocal microscope system

Immunofluorescence staining was performed as described previously^{73 (link)}. Mice were anesthetized with pentobarbital and perfused with 4% paraformaldehyde in PBS at 4 °C for 30 min. DRG were dissected, post-fixed in 4% paraformaldehyde at 4 °C for 2 h, and cryoprotected in 20% sucrose in PBS at 4 °C overnight. Before staining, 10 μm sections were post-fixed with 4% paraformaldehyde in PBS for 10 min and washed with PBST (PBS containing 0.1% TritonX-100). After blocking in 10% goat serum and 0.3% TritonX-100 in PBS for 1 h at room temperature, the sections were incubated at 4 °C overnight with primary antibodies. On the second day, the sections were washed in PBST three times and then incubated with desired secondary antibodies at room temperature for 1 h. The sections were then washed with PBST three times and mounted with Fluoromount-G (Southern biotech, Birmingham, Alabama, USA). Images were obtained using a Leica TCS SP8 confocal microscope system (Wetzlar, Germany).

MG1 was transiently transfected in rice protoplasts to investigate MG1 subcellular localization. Rice protoplasts from 10-day-old ZH11 seedlings were isolated and transfected^{75 (link)}. In brief, rice sheath tissues were cut into 0.5 mm strips and subjected to enzymatic digestion in solution containing 1.5% cellulase R10 and 0.75% Macerozyme R10 for 5.5 h. Then the protoplasts were washed with W5 solution (5 mM KCl, 154 mM NaCl, 125 mM CaCl₂, and 2 mM MES, pH 5.7), and re-suspended in MMG solution (0.6 M Mannitol, 4 mM MES, and 15 mM MgCl₂) to a final concentration of 1.0 × 10⁷ ml^–1. For transfection, 10 µl of plasmids (5–10 µg) was mixed with 100 µl of protoplasts and 110 µl of PEG–CaCl₂ solution (100 mM CaCl₂, 0.6 M Mannitol, and 40% PEG4000). Transfected protoplasts were incubated in W5 solution for 16 h and used for fluorescence observation. To confirm MG1 subcellular localization, the MG1-GFP construct was transiently infiltrated in N. benthamiana leaves as described above. Fluorescence signals were captured with a Leica TCS SP8 confocal microscope system using an excitation laser wavelength of 488 nm with an emission filter of 500–550 nm.

After stimulation, platelets were fixed with 8% PFA 1:1 (v/v) for 15 min at RT before being seeded onto glass coverslips pre-coated with poly-l-lysine for at least 50 min at RT. Attached platelets were washed twice with PBS and then permeabilized with 0.2% Triton X-100 for 10 min. After washing twice, platelets were blocked with 1% BSA o/n at 4 °C. Coverslips were incubated with the primary antibodies (Table S2) for 2 h at RT and washed twice again before incubating with secondary antibodies for 1 h at RT in darkness (Table S2). Phalloidin-iFluor 488 was used for actin detection. Coverslips were mounted with ProLongTM Diamond Antifade Mountant (ThermoFisher Scientific).
Images were captured using a Leica TCS SP8 confocal microscope system with a 100X/1.4 Oil Plan-ApoChromat Ph1 immersion objective and an extra 12X digital zoom. Images were processed with Leica LAS X Software using Lightning process, and analyzed with ImageJ/FIJI Software. Protein colocalization studies were performed using Coloc2 plug-in, analyzing Pearson’s correlation coefficient [7 ]. Protein distribution was analyzed using the Plot Profile plug-in (ImageJ/FIJI software).

Platelets were incubated with 5 μM AF488-labeled fibrinogen (Molecular Probes, F13191) for different times (0, 5, 15, and 30 min) at 37 °C. Then platelets were fixed with 8% paraformaldehyde (PFA) and seeded on poly-l-lysine-treated coverslips. Attached platelets were permeabilized with 0.2% Triton X-100 and blocked with 1% BSA. After labeling with phalloidin-iFluor 647 (Table S2) to visualize actin filaments, preparations were mounted and images were obtained with a Leica TCS SP8 confocal microscope system using a 100x/1.4 Oil Plan-ApoChromat Ph1 immersion objective and an extra 12X digital zoom.

Brain tissue slides acquired from patients with Alzheimer’s disease in different stages of severity, were stained with Thioflavin, DAPI and anti-PML antibodies (as described before). The slides were imaged using the Leica TCS SP8 confocal microscope system with a 100× oil objective (HC PL APO CS2 100×/1.40 Oil) and tile scans (10 × 10 tiles) containing amyloidogenic plaques were recorded. Additionally, several plaque-free regions were captured as control (n = 9 from 3 individuals). PML bodies and cell nuclei were identified and counted using the CellProfiler™ Software. In the tile scans, rectangular areas around the plaques were defined as ‘plaque near’ (n = 31 from 8 individuals) and the surrounding area as ‘plaque distant’ (n = 28 from 7 individuals), not including cells close to the edge of the tile scans. The acquired data from CellProfiler™ were exported to Excel, sorted in those two groups and compared regarding the amount of PML bodies inside the cells and the percentage of cells containing those aggregates.

RAW cells were seeded in a similar way as described to above, on 6-well plates with sterile coverslips at the bottom (n = 2 plates [12 slides]/group). After 24 hours exposure to uPC under starvation, cells were washed with PBS and fixed with 4% FPA. F-actin was stained using phalloidin-TRITC (Hello Bio, HB8621) following the manufacturer’s instructions. Images (n = 6–8 random images/slide) were taken using Leica TCS SP8 confocal microscope system (100× objectives), under the same exposure time.

The N. benthamiana leaves agroinfiltrated with CcCAP1-pBinGFP2, CcCAP1-NLS-pBinGFP2, CcCAP1-NES-pBinGFP2, and the EV control were stained with 5 μg/ml DAPI at 2 dpa. Three hours after staining, the leaves were cut into 8 × 8-mm² pieces and mounted in water on glass slides for confocal microscopy analysis. The fluorescence was imaged using a TCS SP8 confocal microscope system (Leica, Germany). The excitation wavelengths were 488 nm for GFP and 405 nm for DAPI.