After embedded and sectioned, slides were incubated with primary antibody against IL-1β (1:200, R&D Systems, United States), RAGE (1:200, Sigma, United States) and AGEs (1:200, Abcam, Cambridge, MA, United States) at 4°C overnight. Then slides were incubated with biotinylated secondary antibodies and a streptavidin peroxidase complex (PK-7800, Vector Laboratories, Burlingame, CA, United States). Finally, samples observed with microscopy as described previously (Raij et al., 1984 (link); Hong et al., 2019 (link)). The area percentage of the positive staining was calculated with Image Pro Plus 6.0 software (Raij et al., 1984 (link)).
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Immunohistochemical Quantification of IL-1β, RAGE, and AGEs
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