Anti adh1c

Each group of HCC samples was fixed in 10% formalin, embedded in paraffin, and processed as 5 μm continuous sections. Samples were dewaxed with discontinuous concentrations of ethanol and blocked to inhibit endogenous peroxidase. They were then heated in a microwave to retrieve antigens, cooled to room temperature, and then blocked by incubation in goat serum for 30 min at 37°C. Samples were incubated in rabbit anti-ANLN, anti-ENTPD2, anti-TRIP13, anti-PLAC8, anti-G6PD and anti-ADH1C (Abcam, Cambridge, UK; 1:1,200) overnight at 4°C, followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody at 37°C for 30 min and stained using 3,3′-diaminobenzidine. The cell nucleus was stained blue by hematoxylin. Sections were then dehydrated, cleared by xylene, and mounted. ANLN, ENTPD2, TRIP13, PLAC8, G6PD, and ADH1C expression were detected by IHC using a streptavidin peroxidase method. ANLN, ENTPD2, TRIP13, PLAC8, G6PD, and ADH1C expression in the liver were used as a positive control. Samples incubated with PBS instead of the ANLN, ENTPD2, TRIP13, PLAC8, G6PD, and ADH1C primary antibody were used as a negative control. Positive and negative controls were included for each batch of immunohistochemically stained sections. The experimental procedure was performed according to strict adherence to the manufacturers' instructions.

Insulin solution [10 mg/mL], AKT1/2 inhibitor, cycloheximide, actinomycin D, protease inhibitor cocktail, phosphatase inhibitor cocktail I and phosphatase inhibitor cocktail II were purchased from Sigma-Aldrich (St. Louis, MO). Insulin was diluted in basal medium to the necessary concentrations for experimentation as needed. Polybrene [10 mg/mL] was purchased from Millipore (St. Louis, MO) and diluted in medium as needed. Anti-ADH1B, anti-ADH1C, anti-FABP4, and anti-adiponectin antibodies were purchased from Abcam Inc. (Cambridge, MA). Anti-ADH1A was purchased from Origene Technologies, Inc. (Rockville, MD). Anti-DDK, anti-Akt, anti-Phospho-Akt, and anti-PPARγ were purchased from Cell Signaling (Danvers, MA). Anti-GLUT4 was purchased form Bioss, Inc (Boston, MA). Anti-β-actin and IRDye^® secondary antibodies were purchased from LI-COR (Lincoln, NE). All reagents were prepared and stored according to the manufacturer’s instructions.