Dihydroethidium dhe

The level of ROS in the kidney tissue was detected by using the oxidative fluorescent dye dihydroethidium (DHE) (Sigma‒Aldrich, St. Louis, MO, USA). The images were obtained with fluorescence microscopy (Nikon, Tokyo, Japan). The ROS production levels in TCMK-1 cells was measured with an ROS assay kit (S0033M; Beyotime Biotechnology, Shanghai, China). The intensity of ROS was analyzed using ImageJ software.

Macrophages in cell culture were detached in citric saline (0.135 M KCL, 0.015 M Na citrate) while neutrophils were removed by rinsing. Cells were spun down and resuspended in FACS buffer (2% fetal calf serum (FCS; Sigma-Aldrich) in PBS) before incubation with 10μM dihydroethidium (DHE; Sigma-Aldrich) for 30 minutes at 37°C. Fluorescence intensity was measured by flow cytometry with 488nm excitation and 610nm emission.

The excised heart tissue was immediately frozen in liquid nitrogen with optimal cutting temperature compound and sectioned at 10-µm thickness. [24] (link), [25] (link) The section was incubated with 10 µmol/l dihydroethidium (DHE, Sigma Aldrich Co.) at 37°C for 30 min. The fluorescent images were acquired using fluorescence microscope (Olympus IX71, OLYMPUS Optical Co., Tokyo, Japan), and the mean DHE fluorescence intensity of cardiomyocytes, which were in 20 randomly selected fields in each section, was quantitated with Adobe Photoshop CS2 (Adobe Systems Inc.). [24] (link)
Oxidative DNA damage in the myocardium was evaluated by 8-hydroxy-2′- deoxyguanosine (8-OHdG) immunostaining. [26] (link) Heart tissue sections were stained with anti-8-OHdG monoclonal antibody (clone N45.1, Japan Institute for the Control of Aging, Fukuroi, Japan). Briefly, after deparaffinization, the sections were treated with 0.3% H₂O₂ in methanol for 30 min at room temperature, and with 0.1% trypsin for 15 min at 37°C. The sections were then reacted with N45.1 monoclonal antibody (10 µg/ml) for 1 hr at room temperature in a humidity chamber, followed by incubation with DakoEnVision/HRP system (Dako Japan, Tokyo, Japan) for 30 min at room temperature. Sections were then treated with 3, 3′-diaminobenzidine tetrahydrochloride solution (NICHIREI BIOSCIENCES INC., Tokyo, Japan) for 5 min, and counterstaining was carried out with haematoxylin-eosin for 1 min. [27] (link) The positive 8-OHdG nuclei with oxidative DNA damage, which was stained with dark brown, was determined using Adobe Photoshop CS2 (Adobe Systems Inc.), and we calculated the ratio of 8-OHdG positive neclei per total cell number. For this analysis, the digital photomicrographs were taken from 20 random fields at 200× magnification in each section, and the average was obtained from 3 sections.

For the morphological tissue observations using Hematoxylin and Eosin (H & E) staining, some hepatic tissues were fixed with a 10% formaldehyde solution for 24 h. The samples were then exchanged twice with the same solution, dehydrated with double ethanol, and formatted in paraffin, producing a 5 μm thick tissue slice treated with poly-L-lysine. For morphological analysis, the prepared tissue sections were stained with H & E for the liver tissue at ×400 and ×1000 magnification with an optical microscope. The nucleus area was quantified using Image J software version 1.53r (http://rsb.info.nih.gov/ij/ accessed on 15 September 2022) to convert the red intensity from H&E staining.
For oil red O staining, the stained liver samples were then entrenched in Tissue-Tek OCT compound (Thermo, Walldorf, Germany) and frozen. Furthermore, 7 μm sections of these tissues were mounted on 3-aminopropyltriethoxysilane (3-APS)-coated slides and viewed under a Leica microcryotome (model CM1510s, Heidelberg, Germany). Seven successive sectioned slides of each zebrafish were first stained with oil red O (Cat # O0625, Sigma, St. Louis, MO, USA) and then counterstained with hematoxylin, which highlighted the fatty streak lesions. The extent of oxidative stress in these tissues was compared by observing the totality of reactive oxygen species (ROS) with dihydroethidium (DHE, cat # 37291; Sigma, St. Louis, MO, USA) using a Nikon Eclipse TE2000 microscope (Tokyo, Japan), as described elsewhere [66 (link)]. The section fluorescence was quantified using Image J software version 1.53r (http://rsb.info.nih.gov/ij/ accessed on 16 September 2022).
Immunohistochemistry analysis was carried out with the anti-human IL-6 antibody (ab9324, Abcam, London, UK) as the primary antibody and horseradish peroxidase conjugated-anti mouse immunoglobulin G antibody as the secondary antibody using an Envision+ system (K4001, Dako, Denmark).