Levamisole

C. elegans intestines were dissected as described [55 ]. Briefly, adults were transferred to a drop of M9 with levamisole on a microscope slide and the heads and tails were cut with a 25-gauge needle to extract the intestines. Samples were transferred to a microfuge tube and fixed with 4% paraformaldehyde for 1–2 h. FISH was then performed essentially as described for bacteria [56 (link)]. Samples were washed with PBS + 0.1% Tween 20 and then transferred to hybridization buffer (900 mM NaCl, 20 mM Tris [pH 7.5], 0.01% SDS) containing 5 ng/μl probe. Probes were designed against regions of the ribosomal sequence specific to N. parisii and were synthesized with a Quasar 570 (Cy3) 5′ modification and HPLC purified by Biosearch Technologies, Inc. Two N. parisii-specific probes were tested and gave similar results: MicroA (CTCTGTCCATCCTCGGCAA) and MicroB (CTCTCGGCACTCCTTCCTG). These probes also cross-react with the Nematocida sp. 1 sequence isolated from JU1348, the infected C. briggsae strain from India. The universal bacterial probe EUB338 (GCTGCCTCCCGTAGGAGT) was synthesized the same way. Hybridization was performed at 46 °C overnight. Intestines were washed at 48 °C for 1 h in wash buffer (900 mM NaCl, 20 mM Tris [pH 7.5], 0.01% SDS, 5 mM EDTA) and then mounted for microscopy with Vectashield containing DAPI (Vector Laboratories). For Figure 3A–3C, staining was performed in parallel and exposure times were the same for all. FISH staining was also performed as above, except without intestine dissection and with acetone fixation for 15 min at room temperature [57 (link)] instead of paraformaldehyde fixation. These conditions allowed for better staining of spores and were used for the FISH staining shown in Figure 3E and Figure 8G–8I.

The target RNA probe sequences of Cdh2^{42 (link)} and Vangl2^{43 (link)} were amplified by PCR using KOD plus (Toyobo, Osaka, Japan), adenine tailed, and inserted into the pGEM-T easy vector (Promega, Madison, WI). DNA templates were amplified from pGEM-T-Cdh2 or Vangl2 by PCR, using M13 primers and ExTaq (TaKaRa Bio, Kusatsu, Japan). Amplified samples were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). RNA transcription was carried out using the DIG RNA labelling mix (Roche) and T7 or SP6 RNA polymerase (Roche) at 37 °C for 2 h. The products were purified using NucleoSEQ columns (Macherey–Nagel, Düren, Germany) and an equal volume of formamide was added before storage at − 20 °C.
Mouse E8.5 Embryos were fixed in 4% PFA in phosphate buffered saline (PBS) at 4 °C overnight. The embryos were dehydrated using a methanol gradient (25, 50, 75 and 100% methanol) and 1% Tween-20 in PBS (PBT) for a period of 5 min in each solution. After rehydrating samples in a 75, 50 and 25% methanol/PBT gradient, samples were washed with PBT twice. Embryos were bleached with 6% hydrogen peroxide in PBT for 1 h at room temperature followed by washing with PBT thrice. They were subsequently incubated with 20 μg/ml proteinase K in PBT for 6 min at room temperature, followed by post-fixation treatment with PBT containing 4% PFA and 0.2% glutaraldehyde for 20 min and washing with PBT twice. Embryos were next washed with a 1:1 mixture of hybridization solution (50% formamide, 1% SDS, 50 μg/ml yeast tRNA, 50 μg/ml heparin, 5 × SSC, pH 4.5)/PBT and hybridization buffer for 10 min each at room temperature. The embryos were further incubated at 70 °C in hybridization solution for 1 h, followed by replacement of the solution with fresh hybridization solution containing the RNA probe and incubated overnight at 70 °C. The next day embryos were washed with 5 × SSC, pH 4.5 containing 50% formamide and 1% SDS thrice for 30 min each at 70 °C followed by three washes with 2 × SSC, pH 4.5, containing 50% formamide for 30 min each at 65 °C and two washes with RNase buffer containing 0.5 M NaCl, 1% Tween-20 and 0.1 M Tris–Cl, pH 7.5 for 5 min. Subsequently, embryos were incubated with 20 μg/ml RNase A for 30 min at 37 °C and washed thrice with Tris buffered saline containing 1% Tween-20 (TBST) at room temperature. The buffer was then replaced with TBST containing 10% sheep serum and 1% blocking reagent (Roche) for 1 h at room temperature, followed by incubation with anti-digoxigenin-AP Fab fragments (Roche) diluted in a blocking solution overnight at 4 °C. Embryos were then washed thrice with TBST for 5 min at room temperature, five times for 1 h at room temperature, and once overnight at 4 °C. The next day, embryos were washed with 100 mM Tris–Cl, pH 9.5 containing 100 mM NaCl, 1% Tween-20 and 2 mM Levamisole thrice for 5 min at room temperature, followed by replacement of the buffer containing 250 μg/ml NBT and 125 μg/ml BCIP. Whole-mount samples were visualized using a stereomicroscope (SZ9, Olympus, Tokyo, Japan) and images were recorded using a digital camera (DP-50, Olympus). Frozen sections were prepared by embedding stained samples in OCT compound (Sakura Finetek) and immediately freezing on frosted dry ice, followed by perpendicular sectioning against the anterior–posterior axis using a cryostat (CM1850, Leica) set at 10 μm thickness at − 20 °C. The sectioned samples were visualized under an all-in-one microscope BZ-9000 (Keyence, Osaka, Japan).

The collected parasitic L4s and adult H. contortus were thoroughly washed using sterile PBS solution. The samples were anesthetised by 1% levamisole and then the haemozoin-like pigement was observed by a light microscope (Zeiss Primovert iLED, Gttingen, Germany). To identify the ferric iron in the haemozoin, these L4s and adult worms were stained by a ferric iron stain kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, these parasites were fixed with 4% paraformaldehyde for 24 h and stained for 24 h. After the worms were washed and dehydrated, they were carefully transferred to a glass slide and observed using Zeiss light microscopy.

Wild-type and gras-1 hermaphrodites carrying the oxls279[Ppie-1::GFP::H2B, unc +](II); ieSi21 [sun-1p::sun-1::mRuby::sun-1 3’UTR + Cbr-unc-119(+)] IV constructs were grown at 20°C or 25°C and selected at the L4 stage. 14-16h post-L4 live worms were mounted on 2% agarose pads with M9 containing 0.01% levamisole. Hyperstack images (x, y, z, t) at 594nm for SUN-1::mRuby fluorescence, were taken using the 60x or 100X objective at 0.2μm intervals. Nuclei with chromatin in the crescent-shaped configuration characteristic of the leptotene/zygotene stage were imaged every 5 seconds for a minute and SUN-1 aggregate trajectory was followed. An additional stack (x, y, z) capturing GFP::H2B signal at 523nm was collected as a reference for the chromatin shape. Images were registered using the Fiji (NIH) plugin Manual Registration. 2D speed analysis of SUN-1::mRuby aggregates was performed using the Fiji Manual Tracking plugin as in [17 (link),18 (link)].