Pparg

FFPE sections of 4 µm thickness were stained with the following immunohistochemistry (IHC) stains: SCARB1 (1:200; Abcam, Cambridge, UK), PPARG (1:300; Abcam, Cambridge, UK), and adipose differentiation-related protein (ADFP) (1:200; Sigma-Aldrich, St. Louis, MO, USA). IHC staining was performed with a Ventana BenchMark GX (Roche Diagnostics). Antigen retrieval was performed by CC1 antigen retrieval solution (Roche Diagnostics) for 30 minutes. Specimens were incubated with primary antibody for 32 minutes, followed by visualization with the ultraView Universal DAB Detection Kit (Roche Diagnostics) for 12 minutes. Sections were counterstained with hematoxylin, dehydrated, and coverslipped. ADFP is the structural component of lipid droplets, which is required for the formation and maintenance of lipid storage droplets.¹⁰ (link) ADFP immunostaining is helpful in distinguishing sebaceous carcinoma from other neoplasms with overlapping histology.¹¹ (link) Interpretation of SCARB1 staining was done by semiquantitative scoring as follows: negative (0) = 0% to 1% of cells positive, weak (1) = 1% to 25% of cells positive, moderate (2) = 26% to 50% of cells positive, and strong (3) = 51% to 100% of cells positive.¹² (link) Interpretation of PPARG staining was done by semiquantitative scoring as follows: negative (0), weak (1), moderate (2), and strong (3).¹³ (link)

To observe the expression level of osteogenic-related genes and adipogenic-related genes of BMSCs, we conducted immunofluorescence staining according to the standard protocols. First, cells grown on sterile glass coverslips were washed by PBS gently. After fixation by 4% PFA for 30 min at room temperature, the cells were permeabilized by 0.4% Triton X-100 (Biosharp, China) for 30 min and blocked in the presence of 5% normal goat serum (Biosharp, China) for 30 min. Next, the cells were incubated with primary antibodies against RUNX2 (1:1,000; Abcam, UK) or PPARG (1:1,000; Abcam, UK) at 4°C overnight with gentle shaking. Then, the cells were incubated with the specified secondary antibodies for 1 h at room temperature in a dark room. Finally, the nuclei were counterstained with DAPI (Solarbio, China). Images were captured with a fluorescence microscope (Olympus, Japan) equipped with proper filters. Images were captured in 10 randomly chosen fields in each group.