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Fig. 4. Identifying sites by correlative microscopy and in unpermeabilised cells. (A-E) Permeabilised HeLa cells were allowed to extend nascent transcripts in BrUTP, and the resulting BrRNA indirectly immunolabelled with Cy3; cells were then embedded, sectioned (70 nm), and images of the same sections collected by fluorescence and electron microscopy. (A) Merge of fluorescence and –155 eV EM images; sites containing nascent BrRNA (yellow) are scattered throughout the nucleoplasm (grey). Insets, selected areas shown in B-E. (B) Collage of ESI merges (P, red; N, green) of the same region. (C-E) Typical ESI merges (P, red; N, green) of regions shown in insets in A and B. (F-H) Cells were directly fixed, sectioned (without immunolabelling), P and N maps collected and merged, perimeters around N-rich regions mapped (as in Fig. 3C), and nucleoplasmic structures with diameters and P:N ratios typical of active transcription sites (i.e. >40 nm and 0.38±0.1) selected; three examples are shown. These sites by definition have similar diameters and P:N ratios to their labelled counterparts in permeabilised cells; they also have similar textures. Scale bars: 1 µm (A,B); 100 nm (C-H).
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