Fluorescein labeled pna

FISH and WISH were performed as described previously (6 (link), 7 (link), 11 (link), 63 (link), 64 (link)). Parasites were fixed with 4% formaldehyde for 4 h (adults), or 4% formaldehyde containing 1% Nonidet P-40 and 0.2% Triton X-100 for 0.5 to 1 h (cercariae and schistosomula) or 1 to 2 h (juveniles), and then dehydrated in 100% methanol for storage. Five to 10 adult worms (male and female combined) and 5 to 15 juveniles and schistosomula were used for each gene analyzed in each biological replicate. Labeled FISH and WISH probes were generated using either DIG (digoxigenin)-12-UTP (Roche), or fluorescein-12-UTP (Roche). Probes were synthesized by in vitro transcription of partial gene sequences cloned in the plasmid vector pJC53.2, as previously described (63 (link)). Primers used for gene isolation by RT-PCR are listed in SI Appendix, Table S3. Following probe hybridization, specimens were incubated with anti–DIG-AP (MilliporeSigma; 11093274910) for WISH, and anti-DIG-POD (MilliporeSigma; 11207733910) or anti–FITC-POD (MilliporeSigma; 11426346910) for FISH, between 1:1,000 and 1:2,000 dilution, and enzymatic labeling reactions carried out as previously described (64 (link)). Fluorescein-labeled PNA (Vector Labs) was used at 1:500 dilution in a FISH blocking solution overnight at 4 °C (65 (link)).