Fluorescein conjugated avidin

FISH and fiber-FISH were carried out according to published protocols41 (link)61 (link)62 (link). The DNAs labeled with digoxigenin-dUTP (Roche Diagnostics, USA) and Biotin-dUTP (Roche Diagnostics, USA) were detected using rhodamine-conjugated anti-digoxigenin (Roche Diagnostics, USA) and fluorescein-conjugated avidin (Life Technologies, USA), respectively. DNAs were labeled with digoxigenin-dUTP and Biotin-dUTP for FISH analysis. Slides were examined under Olympus BX63 fluorescence microscope (Olympus, Japan). Chromosome and signal images were captured and merged using CellSens Dimension software (Olympus, Japan). Fiber-FISH was conducted to reveal the organization of the centromeric repeat sequences in SES208 genome. The fiber-FISH signals were measured and converted into kb using a 3.21-kb/μm conversion rate41 (link).

Fluorescence in situ hybridization was performed following published protocols (Meng et al., 2018 (link)). First, the chromosome slides were denatured in 70% formamide in 2x SSC at 70°C for 1 min and dehydrated in an ethanol series (70, 90, and 100%; 5 min each). The hybridization mixture (50% formamide, 10% dextran sulfate, 20× SSC, 50 ng labeled probe) was denatured at 90°C for 5 min. Next, the hybridization mixture was applied to the denatured chromosome slides and incubated for 12 h at 37°C. Then, the slides were washed in 2x SSC, 50% formamide in 2x SSC, and in 2x SSC at 42°C for 5 min each. Subsequently, digoxigenin- and biotin-labeled probes were detected using rhodamine-conjugated anti-digoxigenin (Roche Diagnostics, United States) and fluorescein-conjugated avidin (Life Technologies, United States), respectively. Chromosome slides were counterstained with 4′, 6′-diamidino-phenylindole (DAPI) in a VECTASHIELD antifade solution (Vector Laboratories, Burlingame, CA, United States). FISH signals were detected under an Olympus BX63 fluorescence microscope. Images were captured and merged by cellSens Dimension 1.9 software with an Olympus DP80 CCD camera. For image assay, 7–10 cells were analyzed. The final images were processed and adjusted by Adobe Photoshop CC software.