Pv 6001

Paraffin-embedded sections (4-µm thick) were dewaxed and rehydrated using the GTVision™ III anti-rat/rabbit universal immunohistochemical detection kit (cat. no. GK500710; Gene Tech Co., Ltd.) according to the manufacturer's protocol. Antigen retrieval was performed in 0.01 M citrate buffer at 100°C for 6 min. Following cooling, the sections were washed three times in 0.01 M PBS for 5 min each. Subsequently, the sections were incubated with 3% H₂O₂ at room temperature for 10 min. The sections were incubated with a primary polyclonal antibody targeted against NLRP3 (cat. no. ab214185, 1:200; Abcam) at 4°C overnight, and then with a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody for 30 min at room temperature, according to the rabbit polymer detection system (cat. no. PV-6001; OriGene Technologies, Inc.). The sections were stained with DAB reagent (1X) for 3 min at room temperature, counterstained with hematoxylin for 3 min at room temperature and washed by tap water. Stained sections were observed using a Leica DM 6000 B light microscope (Leica Microsystems GmbH). Brown staining indicated a positive reaction. ImageJ software (version 1.8.0; National Institutes of Health) was used for analysis.

HCC tissues and adjacent normal tissues were homogenized in a lysate buffer containing protease-inhibitor (P3480; Merck KGaA, Darmstadt, Germany) and were centrifuged at 6,000 × g at room tempreture for 10 min. Western blot analysis was subsequently performed as previously described (20 (link)). Protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (10 µg/lane) were resolved by 12.5% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Merck KGaA). After blocking with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA), rabbit anti-human AKT1 (ab81283) or GAPDH (ab9485) antibodies (all, 1:2,000; Abcam, Shanghai, China) were incubated with protein samples for 2 h at room temperature. Membranes were washed with PBS for 15 min at room temperature and then incubation with horseradish peroxidase-conjugated polyclonal anti-rabbit immunoglobulin G antibodies (1:10,000; PV-6001; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. Signals were visualized by chemiluminescence detection (Z370398; Merck KGaA). Densitometric quantification of the immunoblot data was performed using Quantity-One software (v3.24; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Immunohistochemical procedures were performed as described previously (21 (link)). HCC tissues and adjacent normal tissues were frozen and coronal sections were cut in a cryostat. The tissues were cut into 4-µm thick sections and mounted on glass slides. The paraffinized sections were heated in an oven at 65° for 24 h, dewaxed to water and rinsed with PBS three times. The washed sections were placed in EDTA buffer (Beinuo Bioscience Inc., Shanghai, China), and then boiled at a low heat following an interval of 10 min at 65°C for a total of 3 intervals. Following natural cooling, the sections were washed with PBS three times, and were placed into 3% hydrogen peroxide solution (Beina Bioscience Inc.), for incubation at room temperature for 10 min, to block endogenous peroxidase. Free-floating sections were rinsed with PBS and placed in a solution containing primary mouse monoclonal antibodies directed against AKT1 (ab81283, 1:2,000; Abcam) at 4°C overnight. After rinsing with PBS, sections were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG mAb (1:5,000 dilution; PV-6001, OriGene Technologies, Inc., Beijing, China). The sections were then washed with PBS and observed by fluorescent video microscopy (BZ-9000; Keyence Corporation, Osaka, Japan).