Figure 4. Immunoreactivity against transient receptor potential vanilloid channel member 5 (TRPV5) and member 6 (TRPV6), respectively is present on the plasma membrane as well as in the cytoplasm of cultured adult human retinal pigment epithelium. Donor cultures (R on the left and U on the right) are the same as those used for western blotting (Figure 2). Cells were plated (passage 146 for R and passage 116 for U) onto uncoated four-well glass chamber slides and grown for 3 days under standard culture conditions (described in the Methods). Monolayers were stained for TRPV5 (upper panels) using an affinity-purified, polyclonal, anti-TRPV5 antibody (8515, final concentration 5 µg/ml). Monolayers were stained for TRPV6 (lower panels) using an affinity-purified, polyclonal, anti-TRPV6 antibody (E-16, final concentration 2 µg/ml). The same secondary antibody was used with both primary antibodies: antigoat Alexa 488 (final dilution 1:500). Nuclei (red) were visualized with TO-PRO-3 (final concentration 2 µM). Scale bars in all panels represent 10 µm. Both plasma membrane (arrows) and prominent, punctuate, cytoplasmic (asterisk) staining is seen for both antibodies.