Analysis of

GPIIb or Fli-1 gene nuclear RNA accumulation and of ploidy level in polyploid megakaryocytes using confocal microscopy. Megakaryocytes were cultured for 8 days and double RNA- and DNA-FISH experiments were performed as in Figure 1A using a GPIIb or a Fli-1 probe (green) and a centromeric probe (red) staining chromosome 12 (Oncor, Gaithersburg, MD). Nuclear DNA was counterstained with DAPI (blue). The ploidy level determined from the number of red spots is indicated as 8N, 16N, or 32N. (A) Representative cells for each ploidy class. Original magnification × 63. (B) Spots corresponding to GPIIb or Fli-1 transcription sites were counted in 15 cells for each ploidy class using a confocal microscope. There were 3 laser excitation wavelengths used: 360 nm, 488 nm, and 543 nm, for DAPI, FITC, and TRITC, respectively. Serial optical sections of 2.5 μm (images collected at 0.25-μm intervals) in the z-axis of the cell were collected sequentially for each marker and overlaid to obtain a 2-dimensional reconstruction. Note that in megakaryocytes with a high ploidy level the spots were counted separately in serial optical sections through the cell. After superposition, 2 or 3 spots could yield a single strong signal. This approach allows a precise counting of the spots.