Anti dnp hrp

As previously described, colorimetric In situ hybridization (ISH) and fluorescent In situ hybridization (FISH) experiments were performed [31 (link)]. Specifically, 10–40 starved (at least four days) worms were killed and stripped of mucus by incubating them for 10 minutes in 7.5% (wt/vol) N-acetyl-L-cysteine (NAC) dissolved in PBS, followed by fixation in 4% (wt/vol) formaldehyde (Sigma-Aldrich #252549) in PBSTx (PBS + 0.3% Triton X-100, Fisher BioReagents, #BP151-500). Worms were stored in 100% methanol at -30°C for a minimum of 16h. Worms were bleached for 3 hours to overnight in formamide-containing solution under bright light, followed by incubation in a proteinase K solution (5 μg/mL proteinase K + 0.1% SDS in PBSTx). For colorimetric ISH, we used digoxigenin-containing (DIG) antisense probes in combination with anti-DIG-AP (alkaline phosphatase) antibody (1:2000, Millipore-Sigma #11093274910). For FISH we used DIG and/or DNP (dinitrophenol) containing probes, detected by tyramide signal amplification using anti-DIG-POD (peroxidase) (1:2000, Millipore-Sigma #11207733910) or anti-DNP-HRP (horseradish peroxidase) (1:2000, Vector laboratories #MB-0603). All samples in each experiment were processed in the same way in a side-by-side manner.

WISH and FISH protocols were modified from previously published methods for planarians (King and Newmark, 2013 (link)) and the mouse bile-duct tapeworm Hymenolepis microstoma (Olson et al., 2018 (link)). Tapeworms were heat killed and fixed in 4% formaldehyde/10% DMSO/1% NP40/PBSTx for 30 min at room temperature before washing and dehydration into methanol. Dehydrated samples were frozen at −30°C for at least 2 days. After rehydration, samples were permeabilized in 10 μg/mL Proteinase-K/0.1% SDS/PBSTx for 30 min, washed into 0.1 M Triethanolamine pH7-8 (TEA), 2.5 μL/mL acetic anhydride was added for 5 min with vigorous swirling, acetic anhydride step was repeated, washed in PBSTx, and post-fixed in 4% formaldehyde/PBSTx for 10 min. Probe synthesis, hybridization, and staining were performed as previously described (King and Newmark, 2013 (link)) using probe concentrations at ~50 ng/mL for 16–48 hr at 56°C. All probes were synthesized with either DIG or DNP haptens and detected using the following antibodies, all at 1:2000: anti-DIG-AP (Sigma), anti-DIG-POD (Sigma), anti-DNP-HRP (Vector Labs). Colorimetric development was done using NBT (Roche)/BCIP (Sigma) or with Fast-Blue (Sigma) (Currie et al., 2016 (link)). Fluorescent signal was visualized after 10–20 min TSA reaction (King and Newmark, 2013 (link)). DAPI staining and mounting were performed as described above.

Single-stranded antisense riboprobes were synthesized with digoxigenin (Dig-11-UTP) (Sigma-Aldrich, St. Louis, MO), fluorescein isothiocyanate (FITC-12-UTP) (Roche, Basel, Switzerland), or 2,4-dinitrophenol (DNP) (PerkinElmer, Inc., Waltham, MA) using standard molecular methods (Collins et al. 2010 (link)). Riboprobes were detected using anti-Dig-POD (1:1000; Sigma-Aldrich, St. Louis, MO), anti-FITC-POD (1:1000; Sigma-Aldrich, St. Louis, MO), or anti-DNP-HRP (1:3000; Vector Laboratories, Newark, CA). Tyramide conjugate signal amplification was performed as previously described (King and Newmark 2013 (link)). The final incubation was with DAPI (10 µg/ml) (1:1000; Thermo Fisher Scientific, Waltham, MA). Animals were mounted in VECTASHIELD (Vector Labs, Burlingame, CA) for imaging.