Tissue microarray analysis of metallothionein and correlation with copper burden. (a) Histogram representation of copper concentrations in the dorsal (blue bars) and ventral (pink bars) liver lobes of individual animals used in the study. The corresponding 2 mm tissue cores punched from formalin-fixed paraffin-embedded sections of the same liver tissue are shown below each bar. (b) Zoomed-in regions of individual tissue cores and the copper and zinc concentrations of the corresponding liver lobes are shown underneath. Briefly, tissue microarray (TMA) slides were dewaxed, re-hydrated and processed according to the protocol described in the Envision dual labelling kit. TMAs were incubated with a 1:250 dilution of anti-MT antibody, in blocking solution (1%(w/v) BSA in TBS pH7.6) overnight at 4°C. TMAs were incubated with a peroxidase-labelled polymer conjugated to goat anti-mouse and goat anti-rabbit immunoglobulins then developed using a DAB buffered substrate. TMA slides were counterstained with haematoxylin, dehydrated and photographed (magnification ×10).