Figure 4

Subnuclear poly(A)+ RNA localizes with discrete foci of masked mRNA in the microspore of M. vestita. a-l, Developing microspores were fixed at 30 minutes of development. a-d, Dual FISH labeling of microspores with (a) 25mer biotinylated probe against SPDS (red) and (b) a poly(T) probe (green). SPDS probe was detected using avidin conjugated TexasRed (red) (a). An avidin/biotin blocking step was performed, followed by labeling and detection of the Poly(T) probe using an avidin conjugated Fluorescein (green) (a). c, DAPI (blue). d, Merge of a-c; inset shows enlarged view of subnuclear poly(A)+ RNA (green) and masked SPDS (red). 25mer biotinylated probes for centrin (red) (e &i) and SPDS (green) (f &j) were used to label sections sequentially. Centrin probe was detected using avidin conjugated TexasRed (red) (e &i). An avidin/biotin blocking step was performed, followed by labeling and detection of SPDS probe using an avidin conjugated Fluorescein (green) (f &j). Sections were counter stained with DAPI (blue) (g &k). Centrin (red), SPDS (green), and DAPI (blue) labeling were merged in h and l, insets show foci of short probe hybridization. Bar = 25 μm.