Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera

Li, Ji Lian; Cornman, R. Scott; Evans, Jay D.; Pettis, Jeffery S.; Zhao, Yan; Murphy, Charles; Peng, Wen Jun; Wu, Jie; Hamilton, Michele; Boncristiani, Humberto F.; Zhou, Liang; Hammond, John; Chen, Yan Ping

The sections were prehybridized in prehybridization solution (50% formamide, 5ϫ SSC [1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 40 g/ml salmon sperm) at 58°C for 2 h and incubated in hybridization buffer with DIG-labeled TRSV probe solution to a concentration of 100 to 200 ng/ml probe in prehybridization solution at 58°C overnight. After hybridization, the sections were washed twice in low-stringency wash solution (2ϫ SSC, 0.1% SDS) at room temperature for 5 min and washed twice in high-stringency wash solution (0.1ϫ SSC, 0.1% SDS) at 52°C for 15 min. The hybridization signals were detected with alkaline phosphatase (AP)-labeled sheep anti-DIG antibody conjugate (Roche Applied Science). The conjugate solution was added to the dry sections and incubated at 4°C for 2 h in a humid chamber. The slides were rinsed three times with washing buffers. The color development was performed by adding the buffer solution containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to the tissue sections and incubating for 3 to 6 h at room temperature with protection from light. The color reaction was stopped by a 5-min wash in Tris-EDTA (0.1 mM, pH 8.0). The nonspecific staining was removed in 95% ethanol overnight. The sections were rehydrated through successive incubation in ethanol (70%, 95%, and 100%) and xylol (twice for 15 min each) and mounted in Eukitt resin. Negative control reactions included regular dUTP instead of DIG-labeled TRSV probe. In situ hybridization slides were observed under a light microscope (Eclipse TE 300; Nikon) and photographed with a Nikon digital camera (DXM 1200). Dark blue coloring indicates where the DIG-labeled probe bound directly to the viral RNA. The section hybridized with the negative control showed pink staining only from the application of nuclear fast red.