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 techniques46 (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 template22 (link). The labelling scheme is reported in the Table2 .
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).
Lymphocyte cell cultures from four buffalo bulls (two river and two swamp) were prepared according to the standard cytogenetic techniques46 (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 template22 (link). The labelling scheme is reported in the Table
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).
Free full text: Click here
