Axioplan 2 imaging

Murine primary enterocytes were isolated from jejunal segments in accordance with the method reported by Haidari et al. [12 (link)]. We infused ³H-cholesterol-containing triolein into C57BL/6J mice by gavage as described above and allowed to absorb the tracer for 3 h. Then, the jejunal segments were excised and cut into 5-mm lengths, placed in ice-cold Matrisperse (BD Biosciences, Franklin Lakes, NJ, USA), and incubated on ice for 4 h. Enterocytes were liberated by agitating mildly, passed through 100-μm nylon mesh, and washed twice in ice-cold PBS. The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% heat-inactivated fetal calf serum (FCS) for up to 2 h. The culture media and cells were recovered at the indicated time points, and ³H radioactivity was counted. We calculated the percentage of cholesterol radioactive tracer in the culture media against the sum of tracer counts in the media and the cell lysates, which was expressed as “cellular efflux (%)”. The purity of the enterocytes obtained was examined by staining for alkaline phosphatase activity using ELF-97 (Life Technologies, Carlsbad, CA, USA), a phosphatase substrate that emits fluorescence upon dephosphorylation. Cells were observed using a fluorescence microscope (Zeiss Axioplan 2 imaging; MOT, Carl Zeiss, Jena, Germany) and images were captured using AxioVision software (Carl Zeiss).

The impact of the tested HCR-FISH protocol parameters on per cell fluorescence was determined by microscopy. Pure cultures, fixed with 70% ethanol or 1% formaldehyde were used. All samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) before observation under an automated epifluorescence microscope (Zeiss Axioplan2 imaging, Carl Zeiss, Oberkochen, Germany) equipped with a monochrome camera (AxioCam MRm, Carl Zeiss Microimaging GmbH, Göttingen, Germany). More than 1000 DAPI stained cells were counted to calculate the relative abundances of targeted groups and all the experiments were performed in duplicates. For relative brightness evaluation the Inspeck Green Microscope Image Intensity Calibration Kit (Molecular Probes, Eugene, Oregon, USA) was used following manufacturer’s instructions. The signal intensity of the FISH positive cells was calculated using the digital image analysis software program ACMEtool2 [53 (link)] on more than 1000 single cells for each experiment. Signal intensity was expressed as relative unit (RU).

To examine whether the morphology of the orbital tissues was damaged by RO injection, we performed RO injection with 0.10 ml of phosphate-buffered saline (PBS) in 5 anesthetized P0 mouse (50 mg/kg pentobarbital), and then eyes were enucleated after euthanization (CO2 inhalation). The samples were placed in a liquid mixture containing 1 % formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and 1.25 % glutaraldehyde (Sigma-Aldrich) for at least 48 h and embedded in paraffin. Serial Sec. (4 μm thick) were cut in a sagittal plane through the cornea, retina, orbital venous sinus and surrounding tissues. The tissue sections were stained with hematoxylin and eosin (HE). Only the sagittal sections, which were cut through the orbital venous sinus and the surrounding tissue, were selected. The images were visualized using a microscope (Zeiss Axioplan 2 Imaging; Zeiss, Oberkochen, Germany).

5×10⁴ isolated PMNs seeded on poly-L-lysine-coated glass coverslips (BD Biosciences) in tissue-culture wells and allowed to settle prior to stimulation as described above. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-MPO (Dako) and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555 and goat anti-rabbit IgG AF488 (Invitrogen). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma) and NETs were visualized using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss).

Detection of reactive oxygen species (ROS) from cellular source was assessed by CellROX green assay (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol. HuREC were loaded with 5 µM CellROX green in culture medium and stained in the dark for 30 min at 37 °C. Stained cells were washed in PBS, mounted using Fluoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) to contemporaneously visualize nuclei. Images were then immediately captured using Zeiss Axioplan-2 Imaging florescence.
Mitochondrial superoxide production was measured using MitoSOX red (ThermoFisher, Waltham, MA, USA), a fluorescent probe targeted to the mitochondria and specific for superoxide. HuREC were loaded with 5 µM MitoSOX red in Hank’s balanced salt solution (HBSS) with calcium and magnesium for 30 min at 37 °C in the dark. Stained cells were then washed and suspended in HBSS, mounted using Fluoroshield mounting medium containing DAPI and immediately analyzed under a Zeiss Axioplan-2 Imaging fluorescence.