Fluoview viewer software

Zebrafish embryos were dechorionated and mounted in 1% low-melting agarose on a 35 mm glass bottomed dish (Asahi Techno Glass) with 0.016% tricaine (Sigma-Aldrich) in fish medium as described previously48 (link). The dish was submerged in fish medium containing 0.001% tricaine. Bone staining with alizarin red s (Wako) was performed as per the manufacturer’s protocol.
Confocal images were obtained with an FV1000 confocal upright microscope system (Olympus) equipped with a 20× water-immersion lens (XLUMPlanFL, NA 1.0). 473 nm and 559 nm laser lines were employed for green fluorescence protein (GFP and Kaede-green), and red fluorescence molecules (mCherry and alizarin red s), respectively. Image files were processed and analyzed using FLUOVIEW Viewer software (Olympus), MetaMorph (Molecular Devices), and Volocity (perkinelmer).

Sections of 5 μm in thickness paraffin block were placed on adhesive-coated slides. Then, sections were deparaffinized with xylene, rehydrated in graded ethanol (from 100 to 70%) and heated for 30 min in a sodium citrate buffer to increase epitope exposure. Additionally, slides were treated with 0.3% (v/v) H₂O₂ in for 5 min, washed with 0.01 M PBS and blocked with 1% BSA, 0.2% Tween 20 in PBS for 1 h at room temperature. Slides were then incubated overnight at 4°C with monoclonal antibody against: Forkhead box P3 (Foxp3 CellSignaling, Danvers, MA, United States), monoclonal antibody against fibroblast surface protein 1, FSP-1 (Abcam, Cambridge, United Kingdom), monoclonal antibody against alpha-smooth muscle actin, α-SMA (Abcam, Cambridge, United Kingdom), and monoclonal antibodies against E-cadherin and N-cadherin (CellSignaling, Danvers, MA, United States). The optimal dilution for all antibodies was 1:100. The reaction antigen-antibody was visualized with avidin-biotin-peroxidase by using 3,3-diaminobenzidine as the chromogen which resulted in brown staining. Slides were counterstained in Harris hematoxylin. Samples were then dehydrated, preserved with a coverslip and reviewed using light microscopy. Pictures were viewed using Olympus FLUOVIEW Viewer software (Tokyo, Japan).

Cells were fixed for 20 min at room temperature with 4% (w/v)
paraformaldehyde in phosphate-buffered saline (PBS) containing 4% (w/v) sucrose.
Cultures were washed with PBS, permeabilized with 0.2% (v/v) Triton X-100 in PBS
for 6 min, again washed in PBS and blocked for 1 h at
room temperature. After labeling with a first primary antibody
(1–3 h at room temperature or overnight at
4 ºC) and washing with PBS, cultures were incubated with
fluorescent secondary antibody conjugated to Alexa Fluor 488, 546 or 633
(1 h at room temperature) and washed with PBS. The cells were
visualized using a spectral confocal microscope (Olympus FV1000, Tokyo, Japan).
Images were captured and digitized using Olympus Fluoview Viewer software. For
some experiments (see Figure 6), cells were
observed with an Olympus Spining Disk (DSU) microscope equipped for TIRF. Images
were captured using a charged-coupled camera (Andor Ixon3, Andor Oxford
Instruments, Oxfordshire, UK). Images were digitized using Olympus Fluoview
Viewer software. In some cases, the images were analyzed using ImageJ (National
Institutes of Health, Rockville, MD, USA) software. All images were processed
using Adobe PhotoShop (Adobe Systems, San Jose, CA, USA).

Confocal microscopy was performed with using a confocal microscope Olympus FV1200 with Tilescan (Olympus, Japan). Images were captured and digitized using Olympus Fluoview Viewer software using a 1024 × 1024 scan format with 20x and 63x objective. All images were processed using Adobe PhotoShop (Adobe Systems, San Jose, CA, USA).

C28/I2 cells were mixed with AG, AG/Col, or AG-Col gels and cultured for 24 h before staining. The cell-gel constructs were fixed with 4% paraformldehyde. After rinsing, the cells were permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 45 min at room temperature, and then incubated with blocking buffer (5% BSA, serum, 20% Polyvinylpyrrolidone (Amresco) in PBS combined into 1:1:1 ratio) overnight at 4 °C. The samples were incubated with primary antibodies against activated β1 integrin (clone HUTS-4; 1:500; Millipore) or total β1 integrin (clone P5D2; 1:100; Santa Cruz Biotechnology) overnight at 4 °C, and then with Alex Fluor 488 anti-mouse IgG (1:1000; Invitrogen) overnight at 4 °C. Finally, cell nuclei were labeled with 4′,6-Diamidino-2-phenylindole (DAPI; Sigma). An Olympus Fluoview FV1000 confocal microscope was used to visualize activated and total β1 integrins and cell nuclei. Images were acquired using a 60× objective lens (1.2 numerical aperture; Olympus). Cells were selected randomly. The fluorescence signal was quantified by measuring average intensity in individual cells using Fluoview Viewer software (Olympus).

Calcium signalling was performed as previously described [19 (link), 22 (link)]. Briefly, cells were grown until ∼80 % confluency on glass-bottomed FluoroDishes (World Precision Instruments, Sarasota, FL, USA). Cells were serum starved for 3 h and pre-treated for 1 h with S1PR1 antagonist (NIBR-0213) [23 (link)] or left in serum-free media. The cells were washed with 1 ml 37 °C HBSS (Invitrogen) supplemented with 20 mM HEPES buffer (Invitrogen) and 5.5 mM glucose (Sigma Aldrich). Cells were then loaded with 2 μM Fluo-8 AM (Invitrogen) in supplemented 37 °C HBSS for 40 min at 37 °C and 5 % CO2. Fluo-8 AM dye was removed and cells were washed with 37 °C supplemented HBSS. Next, cells were left to rest in 1 ml supplemented HBSS at room temperature in the dark for 20 min. Calcium responses were recorded using an Olympus FV1000 Confocal Microscope with ×20 lens. For analysis, images were obtained at a rate of 1 frame/2 s for a total of 250 s. Images were then analysed using the Olympus Fluoview viewer software. Fluorescence was normalised to mean baseline fluorescence (0–30 s) (ΔF/F0). GraphPad Prism 4 software was used to generate calcium response traces presented as ΔF/F0 over time.

HepG2 cells were cultured in glass-bottom cell culture plates at 37 °C for 12 h. After 6 h of starvation with a serum-free medium, cells were overlaid with medium containing R and Q for another 24 h. Following this, the cells were washed with PBS, fixed with paraformaldehyde (4%) for 15 min at room temperature, and permeabilized with Triton X-100 (0.1%) for 5 min, and then cells were blocked in BSA (5% in PBS) for at least 1 h and incubated with antibody against LDLR (1:250 in BSA) overnight at 4 °C. After being washed with PBS three times, cells were immunostained with secondary fluorescence antibody for 1 h at room temperature [52 (link)]. The cell nucleus was visualized with DAPI. After washing with PBS three times, cells were examined using a confocal microscopy, and the fluorescence intensity was analyzed with Fluoview viewer software (Olympus) [47 (link)].