Trpv6

Localization of TRPV6, PMCA1, CTR1, ATP7A, IREG1, and HEPH proteins was examined by immunohistochemistry. Mouse placenta tissues were embedded in paraffin, cut into sections (4 μm thick), deparaffinized in xylene, and hydrated in descending grades of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in TBS-T for 30 min. Nonspecific reactions were blocked by incubating the sections in 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were subsequently incubated overnight at room temperature with antibodies against TRPV6 (1:300, Santa Cruz Biotechnology), PMCA1 (1:300, Swant), CTR1 (1:300, Novusbio), ATP7A (1:300, Santa Cruz Biotechnology), IREG1 (1:300, Abcam), or HEPH (1:300, Abcam) diluted in 1% BSA. After washing with TBS-T, the sections were incubated with a biotinylated secondary antibody (rabbit or mouse IgG, Vector Laboratories) for 1 h at 37 °C and then incubated with ABC Elite (Vector Laboratories) for 30 min at 37 °C. Diaminobenzidine (DAB; Sigma-Aldrich) was used as a chromogen. The sections were counterstained with hematoxylin and mounted in Cytoseal* 60 Mounting Medium (Richard-Allan Scientific Co., Kalamazoo, MI, USA).

siRNA for CBS (hydroxylamine), CSE (dl-propargylglycine), TRPV1, TRPV3, TRPV6, and TRPM4 were purchased from Santa Cruz. H₂S donor NaHS was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Capsaicin and capsazepine were purchased from EMD Millipore (Billerica, MA, USA). The concentrations of Capsaicin and capsazepine used for the treatment were 1 μM.