Rabbit anti hepcidin

After deparaffinization and rehydration, paraffin sections of liver and kidney tissues were incubated with 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity. Then, the sections were subjected to antigen retrieval by heating the sections in a microwave oven in 10 mM sodium citrate buffer (pH 6.0). After blocking with 5% normal goat serum in PBS, liver tissue sections were incubated with rabbit anti-hepcidin (1:200 dilution; Abcam, Cambridge, UK) and kidney tissue sections were incubated with rabbit anti-FPN1 (1:400 dilution; Abcam, Cambridge, UK) at 4°C overnight. After washing with PBS, the sections were incubated with secondary antibodies. Finally, the sections were incubated with H₂O₂-DAB. Negative controls were incubated with 3% serum without primary antibodies. The integrated optical densities (IODs) of hepcidin and FPN1 were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA).

Proteins from liver and kidney tissues were extracted using RIPA buffer with the inhibitor phenylmethanesulfonyl fluoride (Beyotime, Nanjing, China). Protein concentrations were determined using a BCA protein assay kit (Beyotime, Nanjing, China). A total of 40 μg of each protein was loaded into 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billeria, USA). Then, the membranes were blocked for 2 h with 5% skim milk in TBST buffer. The blocked membranes were incubated with rabbit anti-hepcidin (1:500 dilution; Abcam, Cambridge, UK), rabbit anti-FPN1 (1:1000 dilution; Abcam, Cambridge, UK), and rabbit anti-β-actin (1:1000 dilution; Abcam, Cambridge, UK) antibodies at 4°C overnight. After incubation with a secondary antibody conjugated to horseradish peroxidase (1:1000, SantaCruz Biotechnoloy, CA, USA) for 1.5 h at room temperature, proteins were visualized using ECL reagents (Millipore, Billeria, USA).