MITF-independent persister cells regain Mitfa protein expression in recurrent disease. (A) Overview of the multiplex immunohistochemistry protocol. The antibody staining is repeatedly imaged and stripped, and images are pseudocoloured and stacked on ImageJ to generate a composite image. HRP, horseradish peroxidase. (B) DMSO-treated melanoma recurrent disease on top of the head, labelled with Sox10, mCherry and Mitfa antibodies overlaid with Haematoxylin counterstain; white dashed line box indicates area of section shown in B′. Scale bar: 250 μm. (B′) Enlarged views of DMSO-treated tumour section shown in B, showing lack of mCherry staining in melanoma cells positive for Sox10 and Mitfa. Scale bar: 50 μm. Observable mCherry signal represents trapped antibody in loose tissue fragments. (C) (Left, top to bottom) Brightfield images of melanoma tail fin tumour through growth, regression and recurrence. (Right, middle and bottom) Fluorescent images of regressed and recurred melanoma tail fin tumour, showing mCherry fluorescence in the recurred tumour following tamoxifen-mediated recombination in the MRD. Scale bars: 500 μm. (D) Recurrent disease from MITF-independent melanoma persister cells regain Mitfa protein expression. Overview of tamoxifen-treated melanoma recurrent disease in the tail fin, showing Sox10, mCherry and Mitfa protein staining overlaid with Haematoxylin counterstain; white dashed line box indicates area of section shown in D′. Scale bar: 250 μm. (D′) Enlarged views of tumour section shown in D, showing individual staining of Sox10, Mitfa and mCherry proteins. Scale bar: 25 μm. White arrows highlight cells positive for Sox10, Mitfa and mCherry staining. See also Fig. S3.
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