Fitc conjugate anti digoxigenin antibody

Equal amounts of cell cultures, corresponding to 1.5×10 5 cells, collected at the appropriate time points following transfection or infection were processed by on slide FISH assay for the detection of viral nucleic acids, as described (Manaresi et al., 2015) with minor modifications. Cells were spotted on glass slides, fixed in PBS-paraformaldehyde 0.5% at 4 °C for 30 min and permeabilized for 45 min in PBS-saponin 0.2%. Hybridization reaction was carried out in 25 µL of a hybridization solution containing 25 ng of a digoxigenin labeled, random-primed full-length genomic probe (Dig High Prime, Roche). Specimens and hybridization mixture were denatured together by heating at 95 °C for 5 min and then incubated at 37 °C for 12 h. Following hybridization, the slides were washed twice at 37 °C with 50% formamide -2×SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0) buffer and twice at room temperature in 2×SSC buffer, 10 min each. Detection of the hybrids was performed with a FITC-conjugate anti-digoxigenin antibody (Roche) diluted 1:20 in PBS-BSA 1% and incubated for 1 h; after washing in PBS, slides were stained with Evans blue and observed on a fluorescence microscopy (EX 450-490 nm, BA 520 nm).

Aliquots of cell cultures were processed by on slide FISH assay for the detection of viral nucleic acids, as described [30] with minor modifications. Cells were spotted on glass slides, fixed in PBSparaformaldehyde 0.5% at 4°C for 30 min and permeabilized for 45 min in PBS-saponin 0.2%.
Hybridization reaction was carried out in 25 µl of a hybridization solution containing 25 ng of a digoxigenin labeled, random-primed full-length genomic probe (Dig High Prime, Roche).
Specimens and hybridization mixture were denatured together by heating at 95°C for 5 min and then incubated at 37°C for 12 h. Following hybridization, the slides were washed twice at 37°C with 50% formamide -2×SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0) buffer and twice at room temperature in 2×SSC buffer, 10 minutes each. Detection of the hybrids was performed with a FITC-conjugate anti-digoxigenin antibody (Roche) diluted 1:20 in PBS-BSA 1% and incubated for 1 h; after washing in PBS, slides were stained with Evans blue and observed on a fluorescence microscopy (EX 450-490 nm, BA 520 nm).