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

Fig. 8.

Fig. 8.

DNA fragmentation detection by TUNEL assays in female flower anthers. (A–F) Cross-sections of the anther at stage 5 of development. (A) Cross-section of the anther stained with toluidine blue. The anther wall is composed of epidermis, endothecium, middle layer and tapetum. MMCs could be observed at the centre of the anther. (B–F) DNA fragmentation analysis using DAPI staining and TUNEL assays. (B) Cross-section indicating the structure of the anther by light microscopy. (C) DAPI staining evidencing the nuclei of all the anther cells with blue fluorescence. (D) TUNEL assay: green fluorescence indicates nuclei from tapetum and MMCs that are positive for the assay evidencing DNA fragmentation. (E) Positive control of the TUNEL assay: the green fluorescence showing DNA fragmentation is observed in all the anther wall layers together with MMCs. (F) Negative control of the TUNEL assay shows fluorescence corresponding only to the background. (G–L) Cross-sections of anther at stage 7. (G) Cross-section of the anther stained with toluidine blue. The anther wall is composed of epidermis, endothecium, middle layer and tapetum. The MMCs already have a collapsed structure. (H–L) DNA fragmentation analysis using DAPI staining and TUNEL assays. (H) Light microscopy showing the anther structure and the young pollen grains. (I) Blue fluorescence shows the presence of nuclei. (J) All the cells of the anther wall and the MMCs presented DNA degradation signals as evidenced by the green fluorescence in the TUNEL assay. (K) TUNEL-positive control: DNA fragmentation is observed in all the cells of the anther. (L) TUNEL-negative control showing some background fluorescence and autofluorescence in the debris of MMCs. Abbreviations: EP, epidermis; EN, endothecium; TA, tapetum; ML, middle layer. Scale bars: (A, B) = 30 µm; (G, H) = 60 µm.