Successful fluorophore switch in adult zebrafish primary melanoma after tamoxifen treatment. (A) An illustration of the expected fluorophore switch in the primary melanoma tumour upon tamoxifen (T) treatment and imaged 6 days after the start of the treatment. (B) An adult zebrafish with a fully developed primary melanoma tumour (delineated with white dashed line) 2 days before the tamoxifen treatment course (left) and 6 days after the start of the treatment (right) showing de novo expression of mCherry protein in pigmented trunk tumour after tamoxifen treatment (4 µM). N=2 fish, scale bars: 1 mm. (C,D) STD projections of confocal z-stacks of DMSO (0.04%)-treated fish (C, left mCherry, right merged with GFP) and tamoxifen-treated fish (D, left mCherry, right merge) show small clusters of mCherry-expressing cells in the tamoxifen-treated group only (white arrows). N=2 fish, scale bars: 100 µm. (E,F) Paraformaldehyde (PFA)-fixed paraffin-embedded (FFPE) transverse sections of primary melanoma fish (left) stained with Haematoxylin and Eosin, with zoomed images of the tumour (red arrows pointing to the location of the zoomed images). N=2 fish, scale bars: 2.5 mm and 100 µm, respectively. Dashed line boxes indicate area of section shown in E′,F′ and E″,F″. (E′,F′) Immunofluorescence staining of bulk tumour with mCherry antibody (magenta), counterstained with the nuclear marker DAPI (cyan), shows mCherry signal in tamoxifen-treated samples only (F′) compared to DMSO control (E′). N=2 fish, scale bars: 25 µm. (E″,F″) Immunofluorescence staining of invasive tumour shows successful fluorophore switching and mCherry expression in the tamoxifen-treated group only (F″) compared to DMSO control (E″). N=2 fish, scale bars: 25 µm. See also Fig. S1A.
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