Rhodamine conjugated anti digoxin antibody

Multi-color FISH was conducted on meiosis metaphase I chromosomes of parent lines and putative ILs to confirm their chromosomal composition. B. nigra and CentBr (centromeric-specific tandem repeats of Brassica; accession numbers: CW978699 and CW978837, respectively; Wang G. et al., 2011 (link)) DNA were selected as probes and labeled with biotin (dig)-14-dUTP (Roche, Indianapolis, IN) using nick translation with an average length of 500 bp. Slides were prepared following a previously published protocol (Zhong et al., 1996 (link)) with minor modifications. To decompose the cell walls of pollen mother cells, anthers approximately 1–3 mm in length were digested at 37°C for 3 h in an enzyme mixture containing 6% cellulose R-10 and 6.5% pectinase (Sigma, solution in 40% glycerol). Previously described methods (Wang G. et al., 2011 (link)) were followed for FISH analysis. Hybridized probes were visualized using an FITC-conjugated avidin antibody or rhodamine-conjugated anti-digoxin antibody (Roche, Indianapolis, IN). Chromosomes were counterstained using 0.1 mg/mL DAPI (Vector Laboratories, Burlingame, CA). Images of the signals and chromosomes were captured using a CCD camera (QIMAGING, RETIGA-SRV, FAST1394) attached to a Nikon Eclipse 80i epifluorescence microscope (Tokyo, Japan). Image contrast and brightness were adjusted in Adobe Photoshop (8.0).