Novolink polymer detection system 1

Male zebrafish were anesthetized and perfused with PBS containing 4% formaldehyde (EM grade glutaraldehyde solution, Sigma-Aldrich). Brain tissue samples from male zebrafish were fixed with 4% formaldehyde (Sigma-Aldrich) and embedded in paraffin. Brain tissue specimens were cut into 5 μm thick sections using a tissue microtome, and then, sections were mounted on glass slides. Some brain tissue sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich) to assess tissue integrity. Other myocardial tissue sections were subjected to immunohistochemical (IHC) staining with SOD2 (Cat. numbers ab110300; Abcam, Cambridge, UK), tumor necrosis factor (TNF)-α (Cat. numbers #3707; Cell Signaling Technology, Danvers, MA, USA), and caspase-3 (Cat. numbers #9662; Cell Signaling Technology) for 1 h at a room temperature of 25 ℃. By incubating with biotinylated secondary antibody (NovolinkTM Polymer Detection System l, Leica Biosystems Newcastle Ltd., Newcastle, UK) for 30 min and avidin–biotin–horseradish peroxidase (HRP) complex (Novolink™ Polymer Detection System l, Leica Biosystems Newcastle Ltd.) for an additional 30 min. IHC was visualized using DAB Chromogen (NovolinkTM Polymer Detection System 1, Leica Biosystems Newcastle Ltd.), and slides were counterstained with hematoxylin (NovolinkTM Polymer Detection System 1, Leica Biosystems Newcastle Ltd.).

The BALB/c mice were anesthetized and perfused with PBS containing 4% formaldehyde (EM-grade glutaraldehyde solution, Sigma–Aldrich Co.). Heart tissue samples from the BALB/c mice were fixed with 4% formaldehyde (Sigma–Aldrich Co.) and embedded in paraffin. Tissue specimens were cut into sections that were 5 μm thick using a tissue microtome, and then the sections were mounted on glass slides. Some myocardial tissue sections were stained with hematoxylin and eosin (H&E) (Sigma–Aldrich Co.) to assess tissue integrity. Other myocardial tissue sections were subjected to immunohistochemical (IHC) staining with SOD2 (Cat. numbers ab110300; Abcam) and purified rat antimouse tumor necrosis factor (TNF)-α (Cat. numbers #3707; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Incubation with biotinylated secondary antibody (NovolinkTM Polymer Detection System l, Leica Biosystems Newcastle Ltd., Newcastle, UK) for 30 min and avidin–biotin–horseradish peroxidase (HRP) complex (Novolink™ Polymer Detection System l, Leica Biosystems Newcastle Ltd.) for an additional 30 min was carried out. Immunostaining was visualized using DAB Chromogen (NovolinkTM Polymer Detection System 1, Leica Biosystems Newcastle Ltd.), and slides were counterstained with hematoxylin (NovolinkTM Polymer Detection System 1, Leica Biosystems Newcastle Ltd.).