Fv1000 d confocal laser scanning microscope

For the staining of CD39 and CD73, frozen tissue sections were fixed in methanol, and blocked with PBS containing 3% bovine serum albumin (BSA), 20 μg/ml mouse Ig, and 20 μg/ml goat IgG. The sections were incubated with anti-CD39 polyclonal antibodies (pAb) and anti-CD73 pAb, followed by Cy3 F(ab’)₂ anti-guinea pig IgG pAb (Jackson ImmunoResearch) and Alexa Fluor 594 anti-rabbit IgG pAb (Thermo Fisher Scientific), respectively. After being blocked with rat serum, the sections were further incubated with Alexa Fluor 488 anti-PNAd mAb and biotin anti-B220 mAb, followed by Streptavidin-Alexa Fluor 405. Histochemical staining was analyzed using an FV1000-D confocal laser-scanning microscope (Olympus).

HeLa cells were seeded in a BioCoat™ Collagen I Cellware four-well culture slide (Corning, Corning, NY, USA) at a density of 1.0 × 10⁵ cells per well, in 0.5 mL of medium. After overnight incubation, the cells were further incubated for 4 h with fluorescent probes (1.6 × 10⁻² mM) at 34 °C or 40 °C in a humidified atmosphere containing 5% CO₂. After incubation, the cells were rinsed twice with 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS, and 50 μM LysoTracker Red in MEM was added. Cells were then incubated for 30 min at 37 °C. The cells were rinsed twice with 1 mM EDTA in PBS and fixed with 4% paraformaldehyde in PBS for 20 min. After cells were rinsed twice with PBS, coverslips were mounted over cells in VECTORSHIELD^® Hard Set™ Mounting Medium with DAPI (Vector Labolatories, Burlingame, CA, USA). The cells were then observed with a FV1000D confocal laser-scanning microscope (Olympus, Tokyo, Japan).

HeLa cells were grown on coverslips for 24 h and the cells were infected with HPIV3 (MOI = 100); mock-infected cells served as the control. At 48 h post-infection, the cells were washed with PBS before fixation with 3% formalin in PBS at room temperature for 15 min. Cells were washed twice with PBS for 5 min followed by 100% methanol for 10 min at -20°C. Cells were permeabilized with PBS containing 0.05% Triton X-100 for 10 min at room temperature. The cells were incubated with individual primary antibodies for 1 h at room temperature. After being washed twice with PBS, the cells were incubated with secondary antibodies for 1 h at room temperature. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Microscopic imaging was performed with an FV1000-D confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 60x oil-immersion objective.

Confocal laser scanning microscopy (CLSM) observations were conducted with an upright FV1000-D confocal laser scanning microscope (Olympus). For fluorescein diacetate (FDA) and propidium iodide (PI) staining, samples were soaked in dye solution containing 5 μg/ml FDA, 10 μg/ml PI, and 100 mM sorbitol. After 1 min, the samples were observed via CLSM. For calcofluor white staining, samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 60 min under a vacuum at room temperature. The fixed tissues were washed twice for 1 min in PBS and cleared with ClearSee (Kurihara et al., 2015 (link)). The cleared samples were stained with 0.1% calcofluor white in ClearSee solution for 60 min and then washed with ClearSee solution for 30 min. The stained samples were observed via CLSM. UPLSAPO10X or UPLSAPO60XW (Olympus) was used as the objective lens. The excitation wavelength and transmission range for emission were 473 nm and 485 to 560 nm for FDA and green fluorescent protein (GFP), 559 nm and 617 to 717 nm for PI, and 405 nm and 425 to 475 nm for calcofluor white.

For immunofluorescence, HeLa or A549 cells were seeded onto glass cover slips 1 day before transfection. At 24 or 48 h post transfection, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. The cells were then stained with primary antibodies and Alexa Fluor-conjugated secondary antibodies. In the experiments using A3.01 T cells, transfected cells were cultured in round-bottom plates for 48 h. Cells were then fixed with 4% PFA and permeabilized with 0.2% saponin (except for surface GM1 staining), and stained with antibodies. For GM1 staining, fixed cells were stained with Alexafluor594-conjugated Cholera Toxin Subunit B (Thermo Fisher Scientific) for 30 min. Microscopic imaging was performed with an FV1000-D confocal laser scanning microscope (Olympus, Tokyo, Japan). Line plots of the fluorescence intensity were generated by ImageJ software (NIH, Bethesda, MD).
Quantitative analysis of subcellular localization of Gag–GFP was performed as previously reported34 (link)67 (link)68 (link). Briefly, HeLa cells expressing Gag–GFP were fixed and 20 random fields were inspected. Over 100 cells were analysed for the subcellular localization of Gag–GFP, which was either strongly evident at the PM only, at the PM with intracellular accumulations, or diffusely in the cytoplasm.

vSMCs or KDR cells were seeded on a collagen-coated 35-mm glass dishes (Asahi Techno Glass Corporation, Chiba, Japan). After cells firmly attached to the dish, they were washed once with warm PBS and fixed with 4% paraformaldehyde for 5 min at room temperature. Next, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature and then blocked with 1% BSA at 4 °C overnight. The next day, samples were immunostained with primary antibodies (1:1,000 in 1% BSA, 1 h). For secondary reactions, species-matched Alexa Fluor 488- or Alexa Fluor 568-labelled secondary antibody was used (1:1,000 in 1% BSA, 30 min). Just before imaging, the sample was incubated with Alexa Fluor 647-conjugated phalloidin in CGS-Sol A for 1 h. Fluorescence images of EGFP, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 647 were recorded using an Olympus FV1000-D confocal laser scanning microscope (Olympus Corporation) equipped with a cooled charge-coupled device CoolSNAP-HQ camera (Roper Scientific, Tucson, AZ, USA) and a PLAPO × 60 oil-immersion objective lens. To measure the paxillin-positive area, all intensity profiles were analysed using the MetaMorph 7.1.3.0 software.