Rm2125 rtf microtome

Adult Maritigrella crozieri were collected from the Florida Keys (Rawlinson, 2010 (link); Lapraz et al., 2013 (link)). They were fixed in a Petri-dish containing frozen 4% paraformaldehyde (diluted in phosphate buffered saline [PBS]) overnight at 4°C, rinsed in PBS (3 × 5 min, 5 × 1 hr washes) at room temperature and dehydrated in a step-wise ethanol series for histology and immunofluorescence, and in a methanol series for mRNA in situ hybridization. For histology, heads of adult worms (from the pharynx anteriorward) were dissected, cleared in histosol (National Diagnostics), and embedded in paraffin. Paraffin blocks were sectioned at 8–12 µm using a Leica (RM2125 RTF) microtome. Larval stages were fixed in 4% PFA in PBS for 20 min at room temperature, rinsed in PBS for five x 30 min washes and stored in 1% PBS-azide at 4°C for immunofluorescence, or dehydrated into 100% methanol and stored at −20°C for mRNA in situ hybridization.

Embryos were dissected in PBS and pieces including A1 were fixed in PEMFA (4 % formaldehyde in PEM buffer: 100 mM PIPES, 2.0 mM EGTA, 1.0 mM MgSO₄) at room temperature (RT) for 15–30 min, washed in PBT (PBS with 0.1 % Triton-X 100) and stored in ethanol at − 20 °C. Samples were cleared 3 × 10 min in Histosol (National Diagnostics) at RT, infiltrated with paraffin at 60 °C for 2–3 days and hardened in moulds at RT. Sections 6–8 μm thick were prepared on a Leica RM2125RTF microtome. Slides with sections were washed with Histosol, ethanol and re-hydrated to PBT. Slides were placed in a humidified chamber, blocked with 10 % sheep serum (Sigma-Aldrich) in PBT for 30 min at RT and incubated with Phospho-Histone H3 antibody (Invitrogen) diluted 1:130 at 4 °C overnight, Alexa Fluor 568 anti-rabbit secondary antibody (Invitrogen) diluted 1:300 at RT for 2 h and DAPI (Invitrogen) diluted 1:1000. Sections were imaged with a Leica TCS SP5 confocal microscope and photos processed using Fiji (https://fiji.sc).