Fv10i confocal laser scanning microscope

Following the treatments, the cells were fixed in 4% paraformaldehyde in PBS for 10 min followed by washing and permeabilization with 0.1% Triton X-100 PBS for 5 min. Next, samples were blocked with PBS containing 5% fetal bovine serum (FBS) and 1% bovine serum albumin (BSA) for 40 min. After washing, cells were incubated with primary antibodies at 1:100 dilution ratio in PBS with 1% BSA overnight at 4 °C. Primary antibodies used include NANOG (ab80892; Abcam), Sox2 (4744S; Cell Signaling Technology), and SSEA-1 (ab79351; Abcam). The samples were then washed twice in phosphate buffered saline (PBS) with 1% BSA and incubated with secondary antibodies at 1:1,000 dilution ratio in PBS with 1% BSA for 2 h in a light protected environment. Cells were then washed with PBS with 1% BSA prior to mounting in anti-fade media (Vector Laboratories, Inc.). The samples were imaged using FV10i Confocal Laser Scanning Microscope (Olympus, Inc.). Images of the fluorescent cell cultures were then traced in Adobe Photoshop for fluorescence intensity quantification.

[Ca²⁺]f in body-wall muscle cells was observed by assaying expression of the transgene goeIs3[Pmyo-3::GCaMP3.35::unc-54-3′utr, unc-119] (Schwarz et al. 2011 ). For each experiment, 10 worms were analyzed to obtain images of ∼50 muscle cells. GFP fluorescence was observed by using a BX51 fluorescent microscope (Olympus) with a DC73 CCD camera (Olympus) and an FV10i confocal laser-scanning microscope (Olympus). GFP intensity was measured using ImageJ software and the relative intensity was calculated by normalizing the average intensity with the intensity at t = 0.

The muscle tissues were fixed in 4% paraformaldehyde (in phosphate-buffered saline [PBS]) overnight, embedded in paraffin, and cut into 4-μm-thick sections. For the immunostaining of PDGFRα, frozen sections were used. The muscle tissues were mounted on cork disks with tragacanth gum and frozen by immersion in a liquid nitrogen-cooled isopentane bath. The frozen specimens were sectioned at a 7-μm thickness using a cryostat (CM3050 S, Leica Biosystems, Nussloch, Germany). The sections were incubated with blocking solution (Blocking One, Nacalai) for 60 min and subsequently treated with the appropriate primary antibody diluted in blocking solution at 4 degrees overnight. After several washes, the sections were incubated with secondary antibody for 90 min. After several washes, the sections were incubated with secondary antibody for 30 min. The slides were washed several times and mounted with cover slips using VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent images were acquired using an FSX100 Inverted Microscope (Olympus, Tokyo, Japan) and an FV10i confocal laser scanning microscope (Olympus). The contrast of the images was adjusted using Adobe Photoshop CC (San Jose, CA).

GFP-LC3-RFP expressing plasmids were a kind gift from Asst. Prof. Marisa Ponpuak, Department of Microbiology, Faculty of Science, Mahidol University, Thailand. CCA cells were seeded at density of 500,000 cells per well in 60 mm dish and transfected with 1 µg of GFP-LC3-RFP plasmids for 24 h. The next day, the cells were trypsinized and seeded into 8 well chambered slides at 50,000 cells per well density (Lab-Tek, Rochester, NY, USA) and cultured for 24 h. The next day, the cells were treated with ceritinib for 6 h. After washing with warm serum-free medium, the cells were fixed 4% paraformaldehyde + 2% sucrose/PBS for 20 min. Then, the cells were counter-stained with DAPI. Fluorescent image acquisition was performed using FV10i confocal laser scanning microscope (Olympus, Tokyo, Japan).