Lsm5 confocal microscope

BMMs (1 × 10⁴ cells/well) were seeded onto 96-well plates in triplicate, containing 30 ng/mL M-CSF and 50 ng/mL RANKL. After 7 days' culture, 4% PFA was used to fix osteoclasts for 20 min at 37°C [27 (link)]. The rhodamine-conjugated phalloidin (Cytoskeleton, Inc., Denver, CO, USA) was used to detect the cytoskeleton (F-actin ring). BMMs were stained at 37°C for 1 h, and then, washed with PBS three times, each time for 10 min. A LSM5 confocal microscope (magnification, ×10; Carl Zeiss AG, Oberkochen, Germany) was used to observe the F-actin ring. The images were analyzed using the Image-Pro Plus 6.0 software. Sterile bone pieces from the 4 groups were placed into 96-well plates, containing BMMs (1 × 10⁴ cells) with 30 ng/mL M-CSF and 50 ng/mL RANKL. After 12 days, the 0.25% (v/v) trypsin was used to digest the cells on the bone pieces, and the cells were then washed 3 times with PBS. A Quanta 250 scanning electron microscope (SEM; FEI; Thermo Fisher Scientific, Inc.) with a magnification of 10 kV was used to obtain on the bone surface. The resorption area was measured using Image-Pro Plus 6.0.

Procedures for the fluorescent dye FM 1-43 assay were described previously (54 (link)). Wandering third-instar larvae were dissected for body walls in modified HL-3 Ca²⁺-free solution: 70 mM NaCl, 5 mM KCl, 20 mM MgCl₂, 10 mM NaHCO₃, 5 mM trehalose, 115 mM sucrose, and 10 mM Hepes (pH 7.2). For high K⁺ stimulation, the samples were incubated with 4 μM FM1-43 dye (Invitrogen, F35355) for 5 min in modified HL-3 solution containing 90 mM KCl and 1.5 mM CaCl₂ [110 mM NaCl, 90 mM KCl, 10 mM MgCl₂, 1.5 mM CaCl₂, 30 mM sucrose, 5 mM Hepes, 5 mM trehalose, and 10 mM NaHCO₃ (pH 7.2)] and then washed with modified HL-3 Ca²⁺-free solution. The samples were then fixed with 4% PFA for 35 min and washed three times with PBS before mounting for images. Zeiss LSM5 confocal microscope was used to take images with a 40× objective. Fluorescence intensities were calculated with ImageJ software (National Institutes of Health) and normalized to the average loading fluorescence intensity in controls within the same experimental set.

Cells were harvested by incubating with Trypsin-EDTA (Mediatech Inc.) at 37°C for 3–10 min. One hundred μL of cells (~ 2x10⁴ cells) was incubated with 5 μM of dye-labeled affibody or DARPin fusion protein and 1 μM of DARPin-dCK or 2 μM of affibody-dCK for 2 h at 4°C or 37°C. The cells were washed three times in wash buffer containing phosphate buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄), 1% fetal bovine serum (Hyclone), 0.1% sodium azide (Sigma), and resuspended in slide mounting buffer (wash buffer with 20% fetal bovine serum). The cells were then transferred onto glass slides by centrifuging them in a cytospin at 700 rpm for 5 min. Cells were incubated in 100 μL of 4,6-diamidino-2-phenylinodole (DAPI) for 10 min at room temperature, before adding mounting medium (Invitrogen). Cells were imaged in a Zeiss LSM5 confocal microscope and analyzed using software, according to the manufacturer’s protocol. For internalization assays, cells were grown in 24 well plates at 5x10⁴ cells/well in 150 μL, in the presence of 15 μL of CellLight^®Early Endosomes-GFP, BacMam 2.0, CellLight^®Late Endosomes-GFP, BacMam 2.0 and CellLight^®Lysosome-GFP, BacMam 2.0 each (PPC of 30), overnight at 37°C. The cells were then washed and treated with dye-labeled protein (as above), before trypsinization and imaging.

An indirect immunofluorescence assay was performed as described previously [33 (link)]. Mouse anti-HA monoclonal antibody C179, mouse anti-NP monoclonal antibody mAb61A5, rabbit anti-NP polyclonal antibody, sheep anti-NA polyclonal antibody, rabbit anti-M1 polyclonal antibody, and mouse anti-NS1 monoclonal antibody NS1-23-1 were used for viral protein immunostaining. Alexa Fluor 488 conjugated anti-mouse Ig, Alexa Fluor 594 conjugated anti-mouse Ig, Alexa Fluor 594 conjugated anti-rabbit Ig, and Alexa Fluor 488 conjugated anti-sheep Ig (Thermo Fisher Scientific, Waltham, MA, USA) were used for visualization. Specimens were observed using an LSM 5 confocal microscope (Carl Zeiss, Jena, Germany) or a DMI6000 B microscope (Leica Microsystems, Wetzlar, Germany). The Pearson correlation coefficient (PCC) was calculated using the Coloc2 plugin in Fiji/ImageJ.

The construct pGWBB5/DcLCYB2:GFP was transiently expressed in 2-month-old N. benthamiana tobacco leaves by agroinfiltration, as reported previously [20 (link)]. Samples were visualized in a Zeiss LSM5 confocal microscope at 498–525 nm (GFP) and 640–720 nm (Chlorophyll) and processed with the ImageJ software.