Sc 393906

BC and paracancerous tissue microarray samples were obtained from Sinochem Guanghua (Xi'an) Intelligent Biotechnology Co., Ltd. After fixing in 4% paraformaldehyde for 24 h, the specimens were embedded in paraffin and analyzed by immunohistochemical (IHC). The tissue microarray was incubated with SLC27A2 (1:200, sc-393906, Santa Cruz, USA) primary antibody at an appropriate concentration. Images were collected using Caseviewer 2.2.1 software.

Cells were cultured on 15 mm glass-bottom dishes. Cells were starved for 6 h in serum-free medium to assess lipid uptake under intervention or inhibition of SLC27A2. The control group, the shSLC27A2 group, and the drug-treated group were then cultured in DMEM with 2% FBS and 1:100 Lipid Mixture 1 (L0288, Sigma-Aldrich, USA). Either DMSO or Lipofermata was added for 24 h. Prior to fixation with 4% (v/v) paraformaldehyde, the cells were incubated with 1 μM BODIPY 558/568 C-12 (D3835, Invitrogen, USA) for 2 h. The cells were permeabilized with 0.1–0.2% (v/v) TritonX-100 (ST795, Beyotime, China) in PBS for 10 min, followed by washing and blocking with 5% (w/v) bovine serum albumin (BSA) for an additional hour. The fixed cells were incubated with the primary antibody SLC27A2 (1:500, sc-393906, Santa Cruz, USA) overnight at 4 °C. The next day, we washed the cells and incubated them with FITC-labeled secondary antibodies (1:100, Boster, China) for 1 h at ambient temperature. DAPI (C1002, Beyotime, China) was used to stain the nuclei. Subsequently, the images were captured using a fluorescence confocal microscope from Olympus, Japan.

Paraffin-embedded tissue sections and the tissue microarray slide underwent IHC staining. For antigen repair, tissue sections were placed in a repair box containing citric acid antigen repair buffer in a microwave oven, and blocked with 3% hydrogen peroxide for 20 min. Blocking was performed with 5% BSA for 15 min at room temperature. After removing the blocking solution, the slides were incubated with the SLC27A2 primary antibody (1:200, sc-393906, Santa Cruz, USA) at 4 °C overnight. The next day, tissues were covered with secondary antibodies and incubated for 1 h at room temperature. Then, 3,3′-tetrachloride diaminobenzidine was used for color development. Images were obtained using a microscope. In IHC, the percentage of staining tumor cells was calculated by two independent pathologists (0%: 0; < 25%: 1; 25–50%:2; 50–75%: 3; > 75%: 4), and the degree of staining intensity was also recorded (no staining: 0; weak staining: 1; moderate staining: 2; strong staining: 3). IHC staining score = the percentage of stained tumor cells × staining intensity.