Spectrum green dutp

Whole chromosome painting probes for BTA5, BTA12 and BTA23 were obtained by laser microdissection and labelled with Spectrum Orange-dUTP (Abbott Laboratories) by DOP–PCR. Locus specific probes (NANOG—CH240-164H1, CDX2—CH240-301P20, OCT4—CH240-470G19) were obtained from BAC clones (CHORI-240) and labelled with Spectrum Green-dUTP by nick translation (Abbott Laboratories).

Xist and Huwe1 probes were described previously (Chow et al., 2010 (link)). Tsix was detected with a DXPas34 plasmid (Debrand et al., 1999 (link)). Approximately 1 × 10⁵ of F1-21.6 ESCs were plated on gelatin-coated coverslips and incubated for 24 to 48 hr. After fixation and permeabilization, coverslips with cells were washed and stored in 70% EtOH at −20°C. Then the coverslips were dehydrated in 80%, 95%, and 100% EtOH (5 min each) and briefly air-dried. FISH probes were labeled by nick translation (Abbott) with Spectrum Red-dUTP or Spectrum Green-dUTP following the manufacturer's instructions. Labeled probes were precipitated in the presence of salmon sperm (10 μg) and Cot-1 DNA (3 μg), denatured and competed with Cot-1 DNA for 45 min at 37°C. Cells were then directly hybridized with labeled probes at 37°C overnight. Next, coverslips were washed three times in 50% formamide/2 × SSC followed by three washes in 2 × SSC at 42°C. Cells were stained with DAPI (0.2 mg/ml).

The species-specific chromosome painting probes used in this study belonged to the collection of Pauciullo et al.^{22 (link),45 (link)}. In total, six probes were used. Five corresponding to the q-arms of sub-metacentric autosomes (1q, 2q, 3q, 4q and 5q) of river type buffalo (2n = 50) plus the heterosome X that mapped region q21-25.
Lymphocyte cell cultures from four buffalo bulls (two river and two swamp) were prepared according to the standard cytogenetic techniques^{46 (link)}. The buffaloes were karyotyped and were karyologically normal. Therefore, each probe was hybridised on metaphase plates for validation, first individually and then simultaneously with two sequential steps of FISH with three probes each. Only after this preliminary test the probes were used for oocyte analysis.
Each probe was labelled separately with Spectrum Orange-dUTP and Spectrum Green-dUTP (Abbott, USA) in a DOP-PCR reaction using 2 µl of the original probe template^{22 (link)}. The labelling scheme is reported in the Table 2.
Two sequential rounds of hybridisation were performed on the same slide. The whole FISH procedure including probe precipitation, probe and slides denaturation, incubation and washing steps were accomplished according to Pauciullo et al.^{22 (link)}.

CGH and FISH with microsatellite motifs probes were conducted using methods described in our previous study [9] (link), [25] (link). For chromosome probes, we conducted FISH with slight modification. Chromosome probes were labelled by nick translation incorporating SpectrumGreen-dUTP (Abbott, North Chicago, Illinois, USA) or SpectrumOrange-dUTP (Abbott). Each labelled probe was precipitated with 20 µg glycogen as carrier, and dissolved in 15 µl hybridization buffer. The hybridization mixture was placed on a chromosome slide and sealed with a coverslip and rubber cement. Probe DNA and chromosome DNA were denatured by heating the slide on a heat plate at 68.5°C for 5 min. The slides were hybridized overnight in a humid chamber at 37°C. Hybridization was carried out for 2 days in cross-species chromosome painting. The slides were then washed by the following series: 0.4×SSC, 0.3% IGEPAL (Sigma-Aldrich) at 55°C for 2 min followed by 2×SSC, 0.1% IGEPAL at room temperature for 1 min. The slides were dehydrated by ethanol series and air-dried and then counterstained using 20 µg/ml DAPI (4′,6-diamidino-2-phenylindole), 2×SSC and mounted with anti-fade medium, Vectashield (Vector Laboratories, Burlingame, California, USA).

We performed microdissection using an inverted phase-contrast microscope Zeiss Axiovert.A1 (Zeiss, Oberkochen, Germany) equipped with Eppendorf TransferMan NK 2 micromanipulator (Eppendorf, Hamburg, Germany). We prepared glass needles from 1.0 mm diameter capillary glass using a glass capillary puller, Sutter P-30 Micropipette Puller (Sutter Instrument, Novato, Calif., USA) and sterilized using ultraviolet irradiation. We microdissected a W chromosome from freshly prepared slides of a female B. atrox using a glass needle and the micromanipulation system, subsequently transferring the W chromosome into 0.2 ml PCR tubes. The W chromosome DNA (BaW) was amplified using GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, Mo., USA) according to the manufacturer’s protocol with slight modifications according to^{70 (link)}. The volume of the reactions was scaled down to half, and the PCR amplification step was increased to 30 cycles. The W chromosome paint of B. atrox was labeled by nick translation means incorporating SpectrumGreen-dUTP (Abbott, North Chicago, Ill., USA). The hybridization was carried out for 1 day in the B. atrox chromosomes (control) and 3 days in cross-species chromosome painting (Boa constrictor male and female).

We mapped Pogona vitticeps derived 8 BAC clones containing 8 chromosome-linked genes (WAC; KLF6; APTX; CHD1; CTNNB1; TAX1BP1; OPRD1/RCC1 and NR5A1)^{12 (link),19 (link),52 ,53 (link)} to B. constrictor male and female metaphase chromosomes. The clones were selected from Pogona vitticeps genomic BAC library as previously described in Ezaz et al.^{12 (link),52} , Young et al.^{53 (link)} and Deakin et al.^{19 (link)}. All 8 BACs were anchored to P. vitticeps metaphase chromosomes as control (data not shown). BAC DNA was extracted using the Promega Wizard Plus SV Minipreps DNA Purification System following the manufacturer’s protocol, with volumes scaled up for 15 ml cultures. The BACs were labeled with SpectrumOrange-dUTP or SpectrumGreen-dUTP (Abbott, North Chicago, Ill., USA) and hybridized for 2 days. The slides were then washed twice in 0.4 × SSC, 0.3% IGEPAL (Sigma-Aldrich) at 55 °C for 5 min each and after air-dried, counterstained using DAPI (1.2 µg/ml) and mounted in an antifade solution (Vector, Burlingame, CA, USA).