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. 2013 Jul 21;112(5):789–800. doi: 10.1093/aob/mct141

Fig. 7.

Fig. 7.

DNA fragmentation detection by TUNEL assays in male flower anthers. (A–F) Cross-sections of the anther at stage 8 of development. (A) Cross-section of the anther stained with toluidine blue. The anther wall is composed of epidermis, endothecium and tapetum. Tetrads (TET) are still surrounded by callose, and Ubisch bodies (arrow) are released into the locule. (B–F) DNA fragmentation analysis using DAPI staining and TUNEL assays. (B) Cross-section indicating the structure of the anther with light microscopy. The epidermis and endothecium layers are shown. (C) DAPI staining evidencing the nuclei with blue fluorescence. The arrow indicates a nucleus of the endothecium. (D) TUNEL assay: green fluorescence indicates nuclei that are positive for DNA fragmentation; most of them belong to the tapetum (arrows) and some to the endothecium. Note that the nuclei stained with DAPI marked with an arrow in (C) show no DNA degradation. (E) Positive control of the TUNEL assay; the green fluorescence showing DNA fragmentation is observed in all the anther wall layers as expected. (F) Negative control of the TUNEL assay shows no fluorescent nuclei; the observed fluorescence corresponds to the background or autofluorescence since it does not correspond to DAPI staining. (G–L) Cross-sections of anthers at stage 10. (G) Cross-section stained with toluidine blue. The epidermis and endothecium are the only anther wall layers remaining, and tapetum debris is also detected (arrow). Young pollen grains are already developed. (H–L) DNA fragmentation analysis using DAPI staining and TUNEL assays. (H) Light microscopy showing the anther structure and the young pollen grains. (I) DAPI staining evidencing the presence of nuclei with blue florescence. The arrow indicates a nucleus of a young pollen grain. (J) The green fluorescence in the TUNEL assay shows that all the cells of the anther wall presented DNA fragmentation signals; however, the young pollen grain nuclei (evidenced in I) did not present positive signals. (K) TUNEL-positive control: DNA fragmentation is observed in all the anther wall cells and the young pollen grains (arrows). (L) TUNEL-negative control showing some background fluorescence. Abbreviations: EP, epidermis; EN, endothecium; TA, tapetum; TET, tetrads; YPG, young pollen grain. Scale bars: (A, B) = 40 µm; (G, H) = 70 µm.