Mouse monoclonal anti digoxigenin antibody

For the detection of poly(A)⁺ RNA in T. brucei and T. cruzi, the parasites were harvested, washed in PBS (pH 7.4), fixed by incubation in 4% paraformaldehyde and were allowed to adhere to poly-L-lysine-coated slides for 10 minutes. The slides were washed in PBS and the parasites were permeabilized by incubation with 0.2 M HCl (diluted in PBS) for 10 minutes. For T. cruzi, the cells were incubated with prehybridization buffer (35% formamide, 0.02% BSA in 2X SSC buffer) supplemented with 25 µg/ml tRNA, 1 mg/ml salmon sperm DNA (Sigma-Aldrich) and 40 U/ml RNaseOUT (Invitrogen) for 30 minutes at 37°C. For T. brucei, the cells were incubated with prehybridization buffer containing 10X Denhardt’s solution, 1 mM EDTA, 35% formamide in 4X SSC and supplemented with 0.5 µg/ml tRNA and 2 mU/ml RNaseOUT for 30 min at room temperature. Digoxigenin-conjugated oligo(dT) probes (6 ng/µl) were diluted in prehybridization buffer and denatured by heating at 65°C for 3 minutes. Hybridization was performed for 16 hours at 37°C. Probe binding was detected by indirect immunofluorescence analysis with mouse monoclonal anti-digoxigenin antibody (Sigma-Aldrich, 1:300 dilution) and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, 1∶600 dilution), as described previously. As a control, 100 µg/ml RNase A was added to the pretreatment buffer before probe hybridization.