The system is applied to the sections of the communal grazing lands, which are most suitable for grazing.
Then biotinylated anti-mouse/anti-rabbit IgG was applied to the sections, followed by avidin biotin perioxidase complex.
The secondary antibody, a rabbit anti-mouse immunoglobulin (IgG) horseradish conjugate (HRP) (Abcam, Cambridge, UK), was applied to the sections 1 μg/ml for 60 minutes at room temperature.
Secondary antibodies were applied to the sections for 2 hr at room temperature.
Anti-VGF antibody was applied to the sections for overnight at 4°C.
Biotinylated goat anti-mouse IgG and streptavidin conjugated fluorescent 480 (green) were applied to the sections.
Probes were applied to the sections and hybridization was performed in a humidified chamber at 60°C for at least 16 hours.
Subsequently, human nuclei were stained following the protocol previously described and a rabbit polyclonal anti-troponin I primary antibody (Santa Cruz) was applied to the sections.
Briefly, a rabbit antiserum raised against AHS-9 -VP7 crystals was diluted 1/1000 in PBS containing 0.5% bovine serum albumin (PBS-A) and applied to the sections.
Alexa Fluor 488 donkey anti goat IgG (Invitrogen Molecular Probes) was applied to the sections and incubated for 30 minutes at room temperature.
In most cases a minimum of 250 β-globin or Rad23a alleles were analyzed and scored (by a person not knowing the probe combination applied to the sections) as overlapping or nonoverlapping with BACs located elsewhere in the genome.
