Ab128870

The expression of metabolic enzymes in thyroid tumor tissues was assessed via IHC staining using specific antibodies. Successive frozen tissue sections adjacent to the section analyzed using AFAIDESI-MSI were warmed to room temperature for 20 min. Sections were fixed in paraformaldehyde for 10 min. After washing in phosphate-buffered saline, the sections were immersed in 0.25% Triton X-100 for 15 min to make the tissue permeable, and then blocked with 1% bovine serum albumin for 30 min at room temperature. Furthermore, the sections were incubated with antibodies against iPLAs (Proteintech; Chicago, IL, USA; 22030-1-AP; 1:200) and FASN (Abcam; Toronto, ON, Canada, C; ab128870; 1:300) at 4 °C overnight, followed by rewarming at room temperature for 20 min. A PV-9000 two-step IHC kit was used according to the manufacturer’s instructions, and a DAB kit was used to detect antigen–antibody binding (Zhongshan Goldenbridge Biotechnology Ltd. Co., Beijing, China). The slides were counterstained with hematoxylin, dehydrated, mounted, and covered. FASN and iPLAs staining revealed cytoplasmic protein expression. The intensities of FASN and iPLAs were graded semi-quantitatively based on a scale comprising 0 (no staining), low expression (weak-to-moderate staining), and high expression (strong staining).

Total protein was extracted using RIPA buffer (Sigma-Aldrich, USA). Protein concentration was quantified using the Pierce BCA protein assay (Thermo Scientific, USA). Proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride membranes (Millipore, USA). After fixed in 5% skimmed milk, proteins were incubated with primary antibodies overnight at 4 °C. Then, the membranes were washed with PBS buffer, and incubated with goat anti-rabbit IgG H&L (1: 5000, ab96899, Abcam, USA) at room temperature for 1 h. β-actin was selected as the internal reference. Protein bands were visualized using Enhanced Chemiluminescence Detection Kit (Amersham Life Science, USA), and analyzed using Image J software. Primary antibodies were as follows: anti-fatty acid synthase (1:2000, ab128870, Abcam, USA), anti-SCD (1:2000, ab236868, Abcam, USA), anti-ACACA (1:2000, ab269273, USA), anti-ACAT1 (1:2000, ab154396, Abcam, USA), anti-OXSM (1:2000, ab229111, Abcam, USA), anti-VAPA (1:2000, ab176995, Abcam, USA), anti-CBFB (1:2000, ab133600, Abcam, USA) and anti-β-actin (1:2000, ab181092, Abcam, USA).

A total of 132 IMPC and 121 IDC-NOS formalin-fixed and paraffin-embedded (FFPE) tumor tissues were continuously sectioned into 4 μm thickness. Primary antibodies against SREBF1(1:200 dilution, ab191857, Abcam), and FASN (1:3000 dilution, ab128870, Abcam) were used to evaluate protein expression and were reviewed by at least three pathologists. SREBF1 and FASN were graded according to the percentage of positive tumor cells (0 = 0%; 1 < 25%; 2 = 25–50%; 3 > 50%) and the intensity of staining in the tumor (0 = no staining; 1 = weak; 2 = moderate; 3 = high); the two scores were multiplied to obtain an overall score. If the product of the two scores was > 4, they were considered positively stained. And primary antibodies against CD45 (ZM-0183, ZSGB-BIO), CD3 (ZA-0503, ZSGB-BIO), CD8(ZA-0508, ZSGB-BIO), and CD20 (TA800394, ZSGB-BIO) and were also used to independently evaluate protein expression by three pathologists. The immunoreaction was graded as follows: (−) = no positive cells, (+) = 1–25% of the cells stained, (++) = 26–50% of the cells stained and (+++) = 51–100% of the cells stained. The brightfield images were taken on a Leica DMI8 whole-slide scanner at 40X resolution.