Bs 2130r

IHC was conducted according to the protocol of the Two-step IHC Kit (ZSGB-BIO). Briefly, 4 µm thick slides of paraffin-embedded tissue specimens were processed for antigen retrieval with citric acid buffer (0.01 M, pH 6.0), and endogenous peroxidase activity was quenched with 3% hydrogen peroxide solution. Then, the slides were blocked with 10% bovine serum albumin and incubated overnight at 4 °C with the following primary antibodies: anti-MED15 (1:50; 11566-1-AP; Proteintech), anti-SREBP1 (1:100; AF6283; Affinity), anti-SREBP2 (1:100; ab30682; Abcam), anti-FASN (1:50; DF6106; Affinity), anti-ACC1 (1:100; ab45174; Abcam), anti-ACLY (1:100; ab40793; Abcam), anti-SCD1 (1:100; #2794; CST), anti-PLK1 (1:50; DF7004; Affinity), and anti-Ki67 (1:200, BS-2130R; Bioss). The slides were incubated with biotinylated goat anti-rabbit IgG (1:200) for 1 h at room temperature. DAB (ZLI-0918, ZSBio, China) was used to visualize the immune complexes, and the sections were counterstained with hematoxylin. Immunostaining intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD) as previously described [56 (link)].

The animal experiments were approved by the Animal Care and Use Committee of Sun Yat-Sen University. We divided four-week-old BLAB/c nude mice randomly into two groups (n=5), and subcutaneously injected 2×10⁶ control (NC) or AC092171.4 knockdown Huh7 cells. We measured tumor sizes every 4 days and monitored tumor progression using the Xenogen Spectrum small animal imaging system (Caliper, Hopkinton, MA). The mice were sacrificed at 4 weeks after injection. The tumor tissues were harvested and weighed. The tissues were then fixed in formaldehyde solution, sectioned, and subjected to immunohistochemistry using anti-Ki-67 antibodies (1:100; bs-2130R, Bioss, Beijing). To evaluate lung metastasis, we injected 1×10⁶ control (NC) or AC092171.4 knockdown Huh7 cells through the tail vein of nude mice. After six weeks, the mice were sacrificed, and the lung tissues were harvested and fixed in 4% formaldehyde solution. The tumors on the lung surface were counted. Hematoxylin-eosin (H&E) stained sections were evaluated to determine the extent of lung metastases.

Murine liver tissues were collected, fixed in 4% paraformaldehyde for 48 h, dehydrated, and embedded in paraffin. The paraffin tissue blocks were cut into 4 μm-thick sections, deparaffinized with xylene and passed through graded alcohol (10009218, ChengFeng Co., Ltd., Hangzhou, China). To detect the expression of HK2 and Ki-67 in liver tissue, sections were incubated with anti-HK2 antibody (1:200, ab209847, Abcam, Cambridge, UK) or anti-Ki67 antibody (1:200, bs-2130R, Bioss, Beijing, China). Sections were incubated overnight at 4 °C and rinsed 3 times with 1 × PBS for 5 min each time. Reagents were added according to the instructions of the secondary antibody kit (sp-9000; ZSGB-Bio, Beijing, China), and finally DAB (ZLI-9018; ZSGB-Bio, Beijing, China) were added for a 2 min reaction. Four views were randomly collected from each section, and images were acquired using an optical microscope (DM3000, Leica Co., Ltd., Weztlar, Germany).