Histone simple stain kit

Immunohistochemistry was performed using the Histone Simple stain kits (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol concentrations. The sections were treated for 15 min with 3% H₂O₂ in methanol to inactivate endogenous peroxidases and were then incubated at room temperature for 1 h with the primary antibodies against LOX-1 (rabbit anti-LOX-1 antibody, 1:200; Abcam, England) or CD68 (rabbit anti-CD68 antibody, 1:500; Abcam, England). All sections were analyzed using an Olympus B × 40 upright light microscope (Olympus, Tokyo, Japan). For each staining, totally 3 × 7 sections (7 mice) per group were analyzed and the representative images were presented. All image analyses were done by a blinded reviewer.

Mouse heart tissue from each group was stored in 4% paraformaldehyde, embedded in paraffin wax, and cut serially into 4 mm sections. Tissue sections were deparaffinized via immersion in xylene (3 times, for 5 min each) and rehydrated using a descending series of alcohols (100%, 90%, 85%, and 75% alcohol, 5 min each). The sections were stained with hematoxylin and eosin for histological analysis. Biopsy samples were stained using Masson’s trichrome stain to investigate any morphological and fibrotic changes in the heart. Blue staining represented collagen accumulation. Immunohistochemistry was performed using the Histone Simple stain kits (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. The sections were treated for 15 min with 3% H2O2 in methanol to inactivate endogenous peroxidases and were then incubated at room temperature for 1 h with the primary antibodies IL-1, 1:100. The ultrastructure was then observed at 20× magnification using a Leica Microsystems’ CMS GmbH inverted fluorescence microscope (Leica Camera AG, Barnack, Germany).