Dmi600b microscope

In situ ROS production was estimated using the oxidation of the dihydroethidium (DHE) probe to fluorescent products (Davel et al., 2012 (link)). Transverse aortic sections (10 μm) obtained with a cryostat were incubated with PBS at 37°C for 10 min. Fresh PBS containing DHE (5 μM) was topically applied to each tissue section, and the slices were incubated in a light-protected humidified chamber at 37°C for 30 min and then a coverslip was added. Negative control sections were treated with the same volume of PBS as the experimental section but without DHE. Images were obtained with a Leica DMI600B microscope equipped for epifluorescence detection using a 20× objective. ImageJ software (NIH-ImageJ, United States) was used for the quantification.

Images were acquired on a DMI600B microscope (Leica) with an ImagEM EM-CCD Camera (Hamamatsu Photonics, Hamamatsu, Japan) in a spinning disc confocal setup (Yokogawa). Imaging was done using Micro-Manager 1.4 Software (http://www.micro-manager.org). Assembling of frames into a single image was performed in ImageJ.

N2A cells were plated on top of cover slides in 24-well format and transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were fixed in 4% PFA 24 h post-transfection and immunostained using primary anti-V5 mouse monoclonal antibody (R960-25 Invitrogen) and secondary Alexa Fluor 488 goat anti-mouse IgG antibody (A-11001 Invitrogen). Cells were mounted on microscope slides using ProLong Diamond antifade with DAPI (P36962 Invitrogen) and imaged using a Leica DMI600B microscope.

The BrdU Assay was performed according to the manufacturer`s protocol (Roche). 1.5x10⁴ cells/96-well plate were seeded in triplicates in supplemented KBM-Gold medium. After 20 h, cells were serum-starved. 24 h later the medium was replaced by supplemented KBM-Gold with 10 μM BrdU. The incorporation of BrdU was detected after 17 h by anti-BrdU-peroxidase labeled antibody. Enzymatic reaction with TMB (tetramethyl-benzidine) was measured after stopping the reaction with 2 N H₂SO₄ in the Victor II plate reader at 420 nm. For DNA cell cycle analysis, HaCaT pLXSN and HPV8 E6 expressing cells seeded at a cell density of 3x10⁵ in 6-well plates were fixed 4 d later in acetone/methanol for 10 min at -20°C. Cells were treated for 1 h with 1 mg/ml RNase and stained with 5 μg PI per 100 μl cells for 30 min on ice to determine the DNA content by flow cytometry at 488 nm (FACSCanto II, BD Biosciences). The single cell population was gated and cell counts were measured in the PI histogram plot to calculate G0/G1-, S- and G2/M-phases by FACSDiva software 8.0.1 (BD Biosciences). For scratch assay HaCaT monolayers were scratched in 4 cm-dishes. The cells were analyzed at time points 0, 24 and 48 h with Leica DMI600B microscope and Leica application suite software (LAS) V3.06. Cells transfected with p63 siRNA or hsa-miR-203 were scratched 24 h after transfection.

DHE was used to evaluate in situ superoxide generation, as previously described [16 (link),17 (link)]. Nuclear fluorescence captured at 562 nm was quantified using the Image-Pro plus software (MediaCybernetics, Silver Spring, MD, USA) and normalized to cytosolic fluorescence. Aortas were embedded in a freezing medium and transverse sections (10 µm) of frozen arteries were obtained using a cryostat (Company), collected on glass slides, and equilibrated for 10 min in Hanks solution (1.6 mM CaCl₂, 1.0 mM MgSO₄, 145.0 mM NaCl, 5.0 mM KCl, 0.5 mM NaH₂PO₄, 10.0 mM dextrose, 10.0 mM HEPES, pH 7.4) at 37 °C. Fresh Hanks solution containing DHE (2 µM) was topically applied to each tissue section and the slices were incubated in a light-protected humidified chamber at 37 °C for 30 min. Negative control sections received the same volume of Hanks solution in the absence of DHE. Images were obtained with a Leica DMI600B microscope. The number of nuclei labeled with 4,6-diamidino-2-phenylindole (Dapi, positive nuclei) along the vascular wall was automatically counted using Image J software.