Digoxigenin dutp

Whole-mount TUNEL staining was based on a previous protocol [48] (link), with some modifications. After removing the vitelline membrane, embryos were fixed 1 hr in MEMFA, extensively washed in PBS, and stored in ethanol at −20°C. After rehydration in PBS, they were permeabilised in 0,25% Triton X-100/PBS, extensively washed in deionised water, incubated for 1 hr in terminal deoxynucleotidyl transferase (TdT) buffer, and transferred to TdT buffer containing 300 U/ml TdT (Invitrogen) and 1 mM digoxigenin-dUTP (Roche). Incubation was carried out overnight at 23°C. The reaction was stopped in 1 mM EDTA/PBS for 2 hrs at 65°C. Embryos were washed 4 times, 30 minutes each in PBS, and twice in 100 mM maleic acid, 150 mM NaCl, pH 7.5, 5 minutes each, at room temperature. Incubation in blocking reagent and staining with NBT/BCIP were performed as for WMISH [45] .

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).

The genomic DNA of the WT and the mutants were extracted and excised with EcoRI. The DNA probes was amplified and labeled by digoxin using digoxigenin-dUTP (Roche) as shown in Figure 1. The DNA band with the hybridized probe was visualized using an enzyme immunoassay and enzyme catalyzed color reaction with NBT/BCIP (Roche).

The FISH experiments were carried out on specimens ZUEC 17569 and ZUEC 17579
(Table S2),
which represent the populations of Rio de Janeiro and Duque de Caxias,
respectively. The PcP190 satellite DNA sequence previously isolated from
C. gaudichaudii by Vittorazzi et al. (2014 (link)) was amplified to obtain chromosomal probes. For this,
one cloned fragment was amplified by PCR in the presence of digoxigenin-dUTP
(Roche) and primers T7 and SP6, which flank the connection site of the pGEM-T
Easy Vector (Promega). The probes were mixed with salmon DNA (1 ng/μL of probe)
and precipitated in ethanol. The DNA was dissolved in a hybridization buffer at
pH 7 composed of deionized formamide (50%), 2 x SSC, phosphate buffer (40 mM),
Denhardt’s solution, SDS (1%), and dextran sulfate (10%). The in
situ hybridization technique was based on Viegas-Péquignot (1992 ), with modifications for the detection
of digoxigenin-labeled probes with anti-DIG-Rhodamine (Roche).
The microsatellites (CA)₁₅ and (GATA)₈ oligonucleotides
were marked directly with Cy5-fluorochrome at the 5’ end during synthesis
(Sigma-Aldrich) and used as probes in FISH assays that followed the protocol of
Kubat et al. (2008 (link)), under high
stringency (77%) conditions. Images of the hybridized metaphase chromosomes were
captured with an Olympus BX-60 microscope and edited with the Image-Pro Plus
program (Media Cybernetics).

The PAC CTC-820M16, localized in the subtelomeric region of chromosome 14 (14q32.33, Chr14:107106019-107206128 Ensemble draft 75 (ref. 45 (link))) was labelled by nick translation with digoxigenin –dUTP (Roche), using the Abbott Molecular Nick Translation kit, as per manufacturer instructions. The 22-14 alpha satellite probe p14.1 (ref. 70 (link)) was similarly labelled with biotin-dUTP.
For 3D FISH, the transfected cells were incubated in CSK buffer (0.1 M NaCl; 0.3 M Sucrose; 0.003 M MgCl₂; 0.01 M Pipes) for 10 min, and then fixed in 2% formaldehyde/1 × PBS for 5 min. Cells permeabilization was carried out in 0.5% Triton X-100/1 × PBS for 20 min. Following an incubation in 0.1 N HCl for 10 min, and a wash in 2 × SCC, the probes were applied onto the cells, and the probe and nuclear DNA were denatured simultaneously at 85 °C for 5 min. The slides were incubated at 37 °C. The following day, slides were washed three times in 0.1 × SSC at 65 °C.
The probes were detected with anti-digoxigenin antibody, conjugated with rhodamine (Roche), or avidin Alexa Fluor 488 Conjugate (Invitrogen), both at 5 μg ml⁻¹, and the slides mounted in Vectashield DAPI (Oncor). Images were acquired with an Olympus BX-51 epifluorescence microscope coupled to a JAI CVM4+ CCD camera, with Leica Cytovision Genus v7.1.