Immunolocalization of the zinc transporter hZTL1 at the apical membrane of human jejunum and Caco-2 cells

Rabbit anti-hZTL1 antibody was raised against a synthetic peptide corresponding to amino acids 190–211 of hZTL1 (ILSSPSKRGQKGTLIGYSPEGT) and was affinity purified. Human jejunum was obtained from cadaver donors with appropriate ethics committee approval. Frozen sections (7 mm) were fixed for 30 min on ice with 4 % (w/v) paraformaldehyde in PBS. Caco-2 cells, grown for 14 days on polycarbonate filters, were fixed with 100 % methanol for 5 min at room temperature. Sections or cells were permeabilized in 0.1 % (v/v) Triton X-100 in PBS and incubated in blocking solution (5 % w/v BSA, 10 % (v/v) goat serum, 0.1 % Triton X-100 in PBS). After incubation overnight at 4°C with anti-hZTL1 antibody (1:100), slides were washed in PBS and samples were then incubated with FITCconjugated goat anti-rabbit IgG (1:500) for 1 h at room temperature. Caco-2 cells were subsequently treated with propidium iodide to reveal nuclear staining or with a chromogenic alkaline phosphatase stain. Sections or filters were mounted in fluorescence mounting medium under a sealed coverslip and visualized using a Leica confocal microscope.