Fluoview fv1000 microscope

Confocal images of sections co labeled with SOX2 and PAX2, SOX2 and NKX2.2 were taken using FV1000 Olympus Fluoview microscope. Cells showing positive label in the optic nerve were counted over an area of 200 μm at all three stages (E16, P5 and P30) using retina on both sides as a reference. Cells were counted in every third section of through the optic nerve and three different nerves were analyzed for each timepoint.

The TgNAC01 gene lacking the stop codon was cloned in frame into the pK7FWG2 vector with the EGFP gene at the C-terminal portion and named 35S:TgNAC01-EGFP. The pK7FGW2 empty vector, named 35S:EGFP, was used as control. The vectors were used for genetic transformation of thirty-day-old tobacco leaves via Agrobacterium tumefaciens EAH105 strain (Horsch et al., 1984 (link)). Fluorescence visualization was checked in the root differentiation zone of 7-day-old transgenic tobacco lines (T1-first generation) using a FV1000 Olympus FluoView microscope (Olympus, Tokyo, Japan) with an argon laser. Excitation wavelength was calibrated between 488 and 509 nm. Protein subcellular localization prediction was determined with CELLO v.2.5 (http://cello.life.nctu.edu.tw/), WoLF PSORT (https://wolfpsort.hgc.jp/), and LocTree3 (https://rostlab.org/services/loctree3). In addition, HMMTOP (http://www.enzim.hu/hmmtop/index.php) was used for transmembrane prediction.

Cells were seeded in 12-well plate at a density of 1 × 10⁵ cells per well on coverslips overnight. Cells were treated with m-Tyr (150 μg/ml; 24 h), m-Tyr + Phe (150 μg/ml; 24 h) or vehicle, fixed in ice-cold methanol, permeabilized for 10 min with 0.5% Triton X-100/phosphate-buffered saline (PBS), washed twice with PBS and then blocked with 5% bovine serum albumin (BSA) in PBS. Cells were incubated overnight with an anti-STAT3 antibody diluted 1:200 in 3% PBS and 0.1% BSA. Cells were washed with PBS and incubated with fluorescent secondary antibodies Alexa 647 (1:3000) for 2 h. Negative controls were carried out using PBS instead of primary antibodies. Cells were washed, mounted in FluorSave Reagent (Merck Millipore), and imaged by confocal laser scanning microscopy with an Olympus Fluoview FV 1000 microscope (and its software). An Olympus water immersion ×60 objective (1.20 N.A. UPLAN APO) objective was used. Samples were imaged at room temperature. Wide field microscopy was carried on using an Olympus IX71 microscope with an Olympus UApo water immersion ×40 objective (1.15 N.A.), a mercury arc lamp excitation, and suitable filters. Camera: Hamamatsu Orca CCD C4742-95. Samples were imaged at room temperature.

Cells were plated and grown on glass slides for 18 ~ 20 hours and fixed with 4% paraformaldehyde. The slides were then blocked and incubated with the following primary antibodies: Anti-E-cadherin was obtained from Santa Cruz (1:25), Anti-N-cadherin was obtained from Santa Cruz (1:25). Finally, the slides were incubated with fluorescence conjugated secondary antibody (Sigma 1:50) and viewed with a Fluoview FV1000 microscope (Olympus, Japan).

UCC were treated for 72 h with cell line-specific half maximal inhibitory concentration (IC₅₀) doses of PLX before cell lysates were prepared. Immunoblot analysis was performed with whole cell extracts as described in [8 (link)]. Antibodies for target detection and secondary antibodies are listed in Table S1. Targets were visualized by SuperSignal™ West Femto (Thermo Scientific, Rockford, IL, USA) and WesternBright Quantum kit (Biozym, Hessisch Oldendorf, Germany).
Immunocytochemistry was performed as described in detail elsewhere [32 (link)]. Briefly, cells were treated on coverslips with the indicated doses, fixed with formaldehyde, permeabilized, and incubated in blocking solution. Cells were stained by the application of 50 μL of primary antibody solution per coverslip (Table S1) and incubated at 4 °C overnight. After washing, secondary antibodies were likewise added at room temperature for 1 h. After washing, the nuclei were counterstained with DAPI (4′,6-Diamidin-2-phenylindol). Finally, mounting medium (Dako, Santa Clara, CA, USA) was added. Images were taken by means of an Olympus Fluoview FV-1000 microscope (Hamburg, Germany), kindly provided by the Center for Advanced Imaging, Heinrich Heine University (Cai, numerical aperture 1/20, 60× objective). Images were processed using the Fluoview FV-1000 software, version 3.1.

IECs were rinsed with phosphate‐buffered saline (PBS), fixed in precooled 4% paraformaldehyde for 30 min, and blocked with 1% bovine serum albumin at room temperature for 1 h. They were then incubated with primary antibodies against ZO‐1 (1:200 dilution, Cell Signaling Technology) and Occludin (1:300 dilution, Cell Signaling Technology) overnight at 4°C. The following day, cells were treated with secondary fluorescein isothiocyanate‐conjugated goat antirabbit IgG (1:200 dilution, Cell Signaling Technology) in darkness for 1 h. IECs were subsequently stained with 4'‐6‐diamidino‐2‐phenylindole for nuclear visualization and washed thrice with PBS. Images were captured using an Olympus FLUOVIEW FV1000 Microscope.

Cells grown on coverslips were washed with PBS, fixed with p-formaldehyde 4% for 30 min at room temperature, permeabilized, and blocked with Triton x-100 0.1% in PBS with BSA 4% for 30 min at room temperature. Samples were incubated with primary antibodies overnight at 4 °C (HDAC3 1:50 (GeneTex, Irvine, CA, USA), H3K9ac 1:100 (SCBT), H3K9me2 1:100 (GeneTex), Lamin A/C 1:200 (GeneTex) and washed with PBS, followed by incubation with Alexa Fluor™ (Thermo Scientific, Waltham, MA, USA) secondary antibodies for 1 h at room temperature. F-actin was stained with Phalloidin Alexa Fluor™ 488 (Thermo Scientific) and nuclei with DAPI Fluorescent Stain (Thermo Scientific). Coverslips were examined and photographed under a confocal laser-scanning Olympus FluoView™ FV1000 microscope or under the fluorescence microscope Olympus IX81.