Sc 515599

Briefly, the sections had been repaired with the antigen repair solution, observed by using 0.5% Triton X-100 permeabilized, and blocked with 5% (w/v) bovine serum albumin (BSA). Then the sections were incubated with primary antibodies at 4°C overnight. Primary antibodies used were as follows: TGF-β1 (ab215715, Abcam, 1:200), MMP9 (ab58803, Abcam, 1:200), Platelet endothelial cell adhesion molecule-1 (CD31) (M1511-8, Huabio, 1:200), Cleaved caspase-3 (ab179517, Abcam, 1:200), NKG2-D (sc-515599, Santa Cruz, 1:200), and nucleotide-binding oligomerization domain containing 2 (NOD2) (sc-56168, Santa, 1:200). The slides were incubated with secondary antibodies for 60 min. Finally, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (c0060, Solarbio), and the slides were visualized by fluorescence microscopy.

Skin tissue was lysed with RIPA buffer, the protein concentration was measured by BCA protein assay, and equal protein amounts were added electrophoretically separated on precast 4%–20% Precast Gels (Meilun) and then transferred onto PVDF membranes. Following incubation with 5% nonfat milk for 2 h, the membranes were treated with primary antibodies overnight at 4°C. The primary antibodies were used as follows: RelB (10544S, CST, 1:1000), IκBα (ab76429, Abcam, 1:1000), and NKG2-D (sc-515599, Santa Cruz, 1:1000). After washing three times, the corresponding secondary antibody were added and incubated at room temperature for 2 h. Finally, the protein bands were visualized with a Gel Doc imaging system and analyzed with the ImageJ software.