Toluidine blue

For transmission electron microscopy (TEM), juvenile R. lagotis were dissected 5 and 15 h p.e. and fixed in 2.5% glutaraldehyde (Sigma) in complete sterile snail saline (SSS+: 3 mM Hepes, 3.7 mM NaOH, 36 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 4 mM CaCl₂, pH 7.8, 100 mOsm; [5] (link)) at 4°C for 24 h. The specimens were then post–fixed in 1% OsO₄ (Polysciences) in SSS+ for 2 h, washed three times in SSS+, dehydrated in ethanol (50%, 80%, 96%, twice each for 15 min, and 100% three times each for 5 min) and acetone (100%, three times each for 5 min). Subsequently, the tissue was incubated in 100% acetone∶Spurr mixture at increasing Spurr concentrations: 2∶1 for 2 h, 1∶1 for 5 h, 1∶2 for 12 h, followed by pure Spurr resin three times each for 12 h. Then, the material in fresh Spurr resin was transferred to plastic capsules and incubated at 60°C for 48 h. The embedded samples were first sectioned at 2 µm thick sections with a Finesse ME microtome, stained with 1% toluidine blue (Polysciences) and observed under a light microscope (Olympus BX 51). When larvae of T. regenti were detected, 60–70 nm thick sections were prepared using ultramicrotome Ultracut E (Reichert-Jung). These sections were stained with uranyl acetate and lead citrate [22] (link) and evaluated under TEM JEOL 1011 microscope. Digital images were captured using associated software.