Immunohistochemical (IHC) analysis of SREBP-1c and NLRP3 expression

Gingival tissue samples were frozen immediately after surgery in liquid nitrogen and stored at −80°C. Serial paraffin sections of biopsies were cut 5 μm thick, and IHC assays was performed using the immunoperoxidase staining. For inhibition of non-specific binding, the tissue sections were incubated with normal goat serum and non-fat dry milk. Subsequently, the sections were incubated at 4°C with primary antibodies against SREBP-1c and NLRP3, respectively. Sections stained with normal mouse IgG as primary antibody were used as a negative control (Kuo et al., 2016). Sections were then reacted with secondary antibody, followed by incubation with avidin-biotin-peroxidase complex with the addition of DAB (3,3′-diaminobenzidine tetrahydrochloride).