Fig. 5.

Pimonidazole immunostaining is comparable to that of N₃-AZA. (A) Representative micrographs from three independent experiments showing that N₃-AZA click staining and pimonidazole immunostaining overlaps in hypoxic FaDu cells co-treated with both compounds. (B) The micrographs were processed with IMARIS software to quantify channel intensities from N₃-AZA click staining and pimonidazole immunostaining. The ratios of signal (hypoxia):background (normoxia) intensities for cells co-treated with N₃-AZA and pimonidazole [or vehicle control (0.02% DMSO) i.e. columns labelled 0 μM, see Fig. S7 for micrographs of cells not treated with N₃-AZA or pimonidazole] are shown (mean ± S.E.M.). N₃-AZA click staining generated a higher signal to noise ratio compared to pimonidazole immunostaining. (C–F) In vivo comparison of N₃-AZA click staining with pimonidazole immunostaining. Both are concentrated in the same regions of a mouse subcutaneous tumour section (C–E) and of a primary mouse head and neck tumour section (F). Representative micrographs are displayed from at least three independent experiments; scale bar represents 20 μm (A), 1 mm (C–E) and 200 μm (F).