Embryos (S2-S8) were fixed for 4 hours at room temperature in 4% formaldehyde in 1x PBS, followed by 3 x 10 minute washes in 1x PBS and gradual dehydration in 30%, 50%, 70%, 80%, 95% and 100% ethanol. The samples were soaked for 30 minutes in 5% glycerol diluted in 100% ethanol, cleared in xylene for 10 minutes, then soaked in two changes of Clear-rite 3 (Richard-Allan Scientific) for a total of 25 minutes. Paraffin infiltration proceeded with 2 x 45 minute incubations, and embedded embryos underwent serial sectioning (5 µm thickness). Paraffin was removed prior to staining by heating slides at 60˚C for 20 minutes, then performing 3 x 2 minute washes in xylene, 3 x 1 minute washes in 100% ethanol, 3 x 1 minute washes in 80% ethanol before rinsing in tap water. Hematoxylin and eosin staining was performed using the ST Infinity H&E Staining System (Leica Biosystems) in a Leica Autostainer. Slides were incubated for 30 seconds in Hemalast, for 2 minutes in hematoxylin, and were rinsed for 2 minutes in tap water. Next, slides were incubated for 45 seconds in differentiator and 1 minute in bluing agent, with each step followed by a 1 minute tap water rinse, and a 1 minute incubation in 80% ethanol. Slides were stained with eosin for 30 seconds, dehydrated 3 x 1 minute in 100% ethanol and cleared in 3 x 1 minute incubations in xylene.
Whole Mount In Situ Hybridization
Colorimetric and fluorescent WISH was performed as previously described in (King and Newmark, 2013; Pearson et al., 2009), with the following modifications:
Egg capsules for S2-S7 embryos (2-10 days post-egg capsule deposition) were punctured with an insect pin and fixed in 4% formaldehyde in 1x PBS-Triton X (PBSTx) 0.5% for 4-6 hours at room temperature. Fixed embryos were dissected out from the egg capsules, washed in 1x PBSTx (0.5%) for 10 minutes, and subject to incremental dehydration in methanol (10 minutes in 50% methanol, 2 x 10 minutes in 100% methanol). Fixed embryos were stored at -20˚C. S2-S7 embryos were not bleached. Proteinase K treatment was increased to 20 minutes.
S8 embryos (newborn hatchlings, 14-16 days post-egg capsule deposition) were removed from egg capsules prior to fixation. S8 embryos and C4 intact adults (2-4 mm) were incubated in 4% formaldehyde in PBSTx (0.5%) for one hour at room temperature.
S2-S7 embryos were not bleached. S8 embryos and C4 adults were bleached in formamide bleaching solution for 30 minutes to 1 hour under bright light.
S2-3 transplanted hosts and irradiated intact controls (~5-6 mm in length) were fixed for 90 minutes. Worms were bleached for 1.5-2 hours in formamide bleaching solution under bright light.
Sexually mature adult worms (≥ 1 cm in length) were treated with 10% N-acetyl cysteine in 1x PBS for 10 minutes, followed by fixation in 4% formaldehyde in PBSTx (0.5%) for 90 minutes. Worms were rinsed in PBSTx (0.5%) for 10 minutes, and then incubated in 10% SDS in 1x PBS for 10 minutes. Reduction time was increased to 20 minutes. Worms were bleached for 2 hours in formamide bleaching solution under bright light.
Immunostaining was performed after fluorescent WISH development with rabbit polyclonal antibodies against H3S10p (1:1000; Millipore # 06-570), mouse monoclonal antibodies against Smith Antigen (Y12) (1:200, ThermoFisher Scientific, PIMA190490), or mouse monoclonal antibodies against Smed PIWI-1 (1:1000, a generous gift from J. Rink). H3S10p antibodies were detected using preabsorbed Alexa-conjugated secondary antibodies (1:1000, Abcam, ab150086, ab150069, ab150071), while anti-Y12 and anti-PIWI staining was visualized with tyramide development using Goat anti-mouse IgG F(ab')2 HRP (1:1000, Jackson Immunoresearch #115-036-072). Nuclear staining was performed with DAPI (1:5000, 1 mg/mL stock solution, ThermoFisher Scientific, D1306) or with Sytox Green (1:5000, 5 mM stock solution, ThermoFisher Scientific, S7020).
S2-S8 colorimetric and fluorescent WISH samples imaged using light sheet microscopy were mounted as described below, while others were mounted in 80% glycerol supplemented with 2.5% DABCO. C4 and sexual adult samples were mounted in Scale A2 mounting media (Hama et al., 2011).
A Leica M205 FA stereomicroscope was used to capture images of live animals and colorimetric WISH samples. A Leica DM600B upright microscope was used to capture images of histological sections. A Zeiss LSM-510-VIS confocal and a customized light sheet microscope were used to capture Z-stacks for fluorescent WISH samples.
Fixed, stained Smed embryos were mounted in 1% low melt agarose in 1x PBS along with fluorescent conjugated beads required for image registration and reconstruction (FluoSpheres® Polystyrene Microspheres, 1.0 µm, red fluorescent (580/605), Invitrogen/Molecular Probes, F13083; FluoSpheres® Carboxylate Modified Microspheres, 0.1 µm, yellow-green fluorescent (505/515), Invitrogen/Molecular Probes, F8803). 1 µM fluorescent bead stock solutions were diluted 1:10,000 - 1: 360,000, depending on the size of the embryo and the magnification of the detection objective used. Samples were placed in an imaging chamber within a Single Plane Illumination Microscopy (SPIM) system described in (Nakajima et al., 2013). S3-S5 embryos were imaged using either a 10x Plan Apochromat or a 5x Plan NeoFluar objective. Z-stacks were taken every 45˚ around the surface of the samples using a rotating stage, producing 8 stacks of images per embryo. Multiview data sets were reconstructed using Fiji SPIM plugins for data registration and fusion (Preibisch et al., 2010). Completely reconstructed data sets were viewed in the Imaris software package, where they were cropped and masked to remove beads. Cell positions and the embryonic pharynx were marked manually using the 3D Spot Finder function, and the three-dimensional coordinates for marked cells were exported into excel for analysis of cell positions. Colocalization was determined manually on S3-S4 whole embryos, or on crop3D sections (100 µm X 200 µm X 100 µm) of S5 embryos.