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After deparaffinization and hydration, the sections were treated with heated citrate buffer (pH 6.0), 0.3% H 2 O 2, and blocked with 3% BCA successively, then incubated with primary antibody at 4 C overnight and biotinylated secondary antibody

Additionally, the expression of mRNA and protein for both 25-hydroxylases (CYP2R1 and CYP27A1), 1--hydroxylase (CYP27B1), and VDR were downregulated, but that of 24-hydroxylase (CYP24A1) was significantly upregulated in the liver of HFD-fed mice compared to those in controls (Fig