Paraplast plus embedding medium

Test worms were relaxed in cold 2% (v/v) HCl in 5/8 Holtfreter’s solution [27 (link)] for 5 min and then fixed in 4% paraformaldehyde and 30% ethanol in 5/8 Holtfreter’s solution for 3 h at room temperature. The fixed specimens were dehydrated through an ethanol series, cleared in xylene, and embedded in Paraplast Plus embedding medium (Sigma-Aldrich Co., St. Louis, MO, USA). The embedded specimens were cut into 4 μm thick sections and stained with hematoxylin and eosin.

After feeding bioassays, the worms that exhibited the most developed reproductive organs within each group were selected and embedded for histological sectioning. The worms were relaxed in cold 2% (v/v) HCl in 5/8 Holtfreter’s solution⁵⁴ (link) for 5 min and were then fixed in 4% paraformaldehyde and 5% methanol in 5/8 Holtfreter’s solution for 3h at room temperature (RT). The fixed specimens were dehydrated through an ethanol series, cleared in xylene, and embedded in the Paraplast Plus embedding medium (Sigma-Aldrich Co., St. Louis, MO, USA). Embedded specimens were cut into 5 μm thick sections using a microtome (Leica RM2125 RTS, Leica Microsystems Ltd., Wetzlar, Germany). Tissue sections were placed on slide glasses coated with 5 mg/mL egg albumin (Nacalai Tesque, Japan) in 50% glycerin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), stretched at 40°C overnight, and stained with Mayer’s Hematoxylin (No. 3000-2, Muto Pure Chemicals, Tokyo, Japan) and 1% Eosin Y solution (No. 3200-2, Muto Pure Chemicals, Japan).

After the serotonin bioassay, 2 worms from each treatment group were chosen for histological analysis. Individual worms were relaxed in cold 2% (v/v) HCl in 5/8 Holtfreter’s solution for 5 min and then fixed in 4% paraformaldehyde and 5% methanol in 5/8 Holtfreter’s solution for 3 h at room temperature. The fixed specimens were dehydrated through an ethanol series, cleared in xylene, and embedded in Paraplast Plus embedding medium (Sigma-Aldrich Co., St. Louis, MO, USA). The embedded specimens were cut into 4-µm thick sections and stained with hematoxylin and eosin (HE) using Mayer’s Hematoxylin Solution (Wako, Osaka, Japan) and Eosin Y (yellowish), Certistain® for microscopy (Merck, Darmstadt, Germany). Specimens were examined, and images were taken using a digital microscopy setup involving a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) and an Olympus DP22 digital camera (Olympus Corporation).