Fig. 2: Establishing Oligopaints to label individual chromocenters in Arabidopsis.

a Schematic illustration of the branched-DNA amplification of Oligopaints signals. Each FISH probe comprises a 36-42-nt sequence (black) complementary to the target DNA, a reverse index (light gray), a reverse transcription primer sequence (dark gray), and a handle sequence (green) containing the binding site for a preamplifier. The preamplifier contains four tandem annealing sites for an amplifier, which can recruit four fluorescently labeled oligos. The branched-DNA amplification design could result in a 32-fold multiplexed amplification of the FISH signal. Three sets of the handle, preamplifier, amplifier, and fluorescent oligo sequences were designed to allow the simultaneous labeling of three DNA targets with different fluorophores (see “Methods”). b Diagram of the five chromosomes in Arabidopsis, depicting the target sites of the CEN178, KEE7, and pericentromeric FISH probes. c Confocal Oligopaints images showing co-labeled CEN178 CC signals with chromosomal-specific pericentromeric probes in pavement cell nuclei from the cotyledons of 4-d-old PBC seedlings grown under 10 µmol m⁻² s⁻¹ R light at 16 °C. These are representative images of at least 60 images for each FISH experiment. The boundaries of the nucleus and nucleolus are traced with dashed blue and orange lines, respectively. Scale bars are equal to 2 μm. d Oligopaints probes and their fluorophore colors used for simultaneously labeling ten CCs in a single nucleus in (e). e Confocal Oligopaints image showing ten CCs labeled with the combinations of three fluorophores depicted in (d). The boundary of the nucleus is traced with a dashed blue line. The scale bar equals 2 μm. This is a representative image of at least 30 FISH images with similar results.