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Fig. 6. (a-d) Immunolocalisation of nascent RNA after pulse labelling with FU and (e) total RNA staining with SYTO RNASelect in unfixed macronuclei. Transcription occurs in distinct foci colocalised with nucleoli and dispersed chromatin. Mid optical section of one macronucleus (a-c). Fluorochrome channels were scanned sequentially generating 8-bit greyscale images. (a) FU incorporated into newly synthesised RNA. (b) To-Pro-3. (c) Overlay, with false colours assigned to each channel (FU, red; To-Pro-3, blue). (d) Average intensity projection of 62 serial optical sections to a plane between XZ-coordinate axes and 3D reconstruction of the nuclear surface (blue) with one optical section. FU incorporated into newly synthesised RNA (green). Putative nucleoli are marked by a red line, domains of dispersed chromatin are marked by a yellow line. The surface was partly cut to look inside. (e) The rear zone of the replication band remains unstained when SYTO RNASelect is used as a RNA selective dye (green).
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