We find that, when the same antibody is used in successive CycIF cycles, signal intensity is highly correlated and morphology retained, as illustrated by p-ERK, p-S6S240/244, Ki-67 and PCNA in BRAFV600E melanoma cells treated with the Raf-specific kinase inhibitor vemurafenib to create samples in which levels of ERK and S6 phosphorylation vary in a predictable manner (Fig. 1e, Supplementary Figs 5 and 6). The gene discussed is MKI67; the disease is melanoma.