Nis elements software version ar 3

H9c2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS at 5% CO₂ and 37°C in an incubator. To determine cell size, cells were seeded on coverslips at a density of 1 × 10⁴ cells/well, serum‐starved overnight, and pretreated with vehicle or syringic acid (10 μM) 3 h before isoproterenol stimulation. Next, the cells were co‐treated with isoproterenol (10 μM) and syringic acid for 24 h. Cells were fixed with 3.7% paraformaldehyde for 30 min, washed twice with phosphate‐buffered saline, permeabilized with 0.2% Triton X‐100 and incubated with Alexa Fluor 488 phalloidin (1:200) for 45 min. The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole, and the samples were sealed with a glass slide. The cell size was measured using NIS Elements Software Version AR 3.0 (https://www.nikonmetrology.com/images/brochures/nis‐elements‐en.pdf, Nikon, Tokyo, Japan).

OIR was performed and quantified as described by Connor et al.60 (link). C57BL/6 mice pups along with dams were exposed to 75% oxygen from P7 to P12 and at P12 they returned to room air to develop the relative hypoxia. Mice pups of the same age kept at room air were used as controls. At P17 mice pups were sacrificed, eyes were enucleated and fixed in 4% (w/v) paraformaldehyde for 1 hr at room temperature. Retinas were isolated, stained with isolectin B4, flat mounts were made, coverslips were placed and examined under a Zeiss inverted fluorescence microscope (Zeiss Observer. Z1). Retinal vasculature was quantified by calculating the ratio of fluorescence intensity to the total retinal area. Retinal neovascularization was measured by first setting a scale with a tolerance point of 50 pixels based on the fluorescence intensity in the screenshot using Nikon NIS-Elements software version AR 3.1. Neovascularity (values above 50 pixels tolerance set point) was highlighted in red and then quantified by dividing the fluorescence intensity in the highlighted area by the total fluorescence intensity in the screenshot (n = 6 eyes).

Cryo-sections were immunostained with utrophin (1:50) alone, or double-immunostained with collagen type I (1:100) and cthrc1 (1:50) antibodies. Cell nuclei were stained with DAPI (1:1000). As secondary antibodies goat anti-mouse IgG antibodies with Alexa Fluor dye were used for collagen type I detection and Cy3 anti-goat for utrophin detection. Microscope observations and image acquisition were performed with the Olympus IX 81 inverted laser scanning confocal microscope (Fluoview 500; Olympus, Tokyo, Japan), equipped with a 40 -nm diode laser, a 488-nm argon-ion laser, a 543-nm helium-neon laser, and a 60 × 1.0 NA PlanApo water-immersion objective. The transmitted-light images were obtained by means of Nomarski differential interference contrast. The Z-stacks were each 1.2 μm each and extended 8 μm into the tissue. “GREEN” was excited at 488 nm and the emission was collected through a BA 505–525 filter; “RED” was excited at 543 nm and the emission was collected through a BA 560 IF filter. The 3-D reconstruction of the utrophin, collagen type I and cthrc1 levels was prepared by Nis-Elements AR software version 3.2 (Nikon Instruments Inc., Melville, NY, USA), and their levels were evaluated by NIS-Elements microscope imaging software (Nikon Instruments Inc).

For the immunocytochemical detection of SEC62 and SOX2 in UM-SCC1 cells, slides from UM-SCC1 cells suspended in 20 ml PreservCyt solution were prepared using the ThinPrep^®-system (Hologic Deutschland GmbH, Wiesbaden, Germany) and dried for 30 min at room temperature. Following rehydration in distilled water and a three-fold wash step in phosphate buffered saline (PBS), samples were fixed in formalin for 15 minutes. Next, epitope unmasking was performed by incubation with Target Retrieval Solution (Dako GmbH, Glostrup, Denmark) at 95°C for 30 min. After cooling to room temperature and three consecutive washing steps with PBS, unspecific binding sites were blocked with 3% BSA (bovine serum albumin) in PBS and the slides were incubated with the primary antibody (1:400 dilution in 3% BSA/PBS) for 30 min at room temperature. The same antibodies as described for western blot were used. Visualization was performed using the Dako REAL^™ Detection System Alkaline Phosphatase/RED (Dako GmbH; Glostrup, Denmark) according to the manufacturer's instructions. Finally, the slides were counterstained with hematoxylin. Each analysis included negative controls by omission of the primary antibody. Slides were imaged using the Nikon Eclipse TE2000-S inverted microscope, Nikon Digital Sight DS-5Mc camera and NIS-Elements AR software version 3.2 (Nikon; Tokyo, Japan).

Cell migration was analyzed using the BD Falcon FluoroBlok system (BD, Franklin Lakes, NJ, USA) with 8-μm pore inserts for 24-well plates. 5 × 10⁴ UM-SCC1 cells transfected with either siRNA or plasmids were loaded into the inserts in normal medium containing 1% FBS. The inserts were then placed in the wells of a 24-well plate in medium with either 10% FBS as a chemoattractant for migration or without FBS (negative control). After 72 h, the cells were fixed with methanol, the nuclei were counterstained with DAPI and the number of migrated cells was analyzed using a bottom reading fluorescence microscope. Three representative images of each insert were obtained, and the number of migrated cells was quantified using NIS-Elements AR software version 3.2 (Nikon, Tokyo, Japan). All cell migration experiments were repeated three-fold (n = 3), and a triplicate of every cell population was analyzed in each experiment.

HUVEC cells were counted, pelleted and resuspended in phenol red-free medium containing 5% DCC-stripped serum; in F-12 K (a positive control containing Endothelial Cell Growth Supplement, BD Biosciences #356006); or in 48 hr-conditioned media from NMFs or BJ3Z cells grown in phenol red-free medium and 5% DCC-stripped serum. HUVEC were seeded in duplicate into 8-well chambers (40,000 cells per well) pre-coated with Matrigel. Cells were incubated for 24 h, with images captured every 2 hours. Cells were photographed in a Nikon Eclipse Ti microscope coupled to a Nikon digital camera DS-Qi1Mc. Images were analyzed and quantified in NIS-Elements AR software version 3.2 (Nikon Corp.). For quantification, the lengths of ten tubular structures/field were measured in duplicate per condition at 6 h of incubation. The experiment was repeated three independent times with similar results.