Scanning electron microscope

Frozen aortic root sections adjacent to the sites of the maximum lesion area were stained with primary antibodies against CD68 (marker for macrophages, rabbit polyclonal; Abcam, catalog number ab125212), and terminal ileum tissues were stained with antibodies of zonula occludens-protein 1 (ZO-1, 33-1500, Thermo Fisher Scientific), Occludin (OCLN, 339188, Thermo Fisher Scientific), toll like receptor 4 (TLR-4, sc-293072, Santa Cruz), and toll like receptor 9 (TLR9, sc-47723, Santa Cruz) respectively, followed by their corresponding secondary antibody conjugated to horseradish peroxidase (HRP, SV0002 or SV0001, BOSTER, Wuhan, China). Staining was illuminated by Diaminobenzidine (DAB), and pictures were taken by a Leica scanning electron microscope and analyzed using Image J software.

A resorption pit assay was employed to test the osteoclastic absorption function. BMMs were plated at a density of 1 × 10⁴ cells/well in 48-well plants with Osteo Assay Plates (Yaoang Biological Technology Co., Ltd., Shanghai, China). Subsequently, samples were treated with Uro-A (0, 5, and 10 μM) and 50 ng/ml RANKL and M-CSF to generate mature osteoclasts as mentioned above. After 5 days, a scanning electron microscope (Leica, Germany) was employed to observe the morphology of osteoclasts and absorption area during differentiation.