Detailed Schistosoma Morphological Analysis

The adult worms recovered after 37 days of mouse infection were fixed and stored in Alcohol-Formalin-Acetic Acid (AFA, 95% ethanol, 3% formaldehyde, and 2% glacial acetic acid), at room temperature, and stained with 2.5% chloride carmine, dehydrated in alcoholic series (70, 90%, and absolute), clarified in methyl salicylate with Canadian balsam (1:2), and prepared as whole-mounts (Neves et al., 1998 (link)). We analyzed at least six males and six females recovered from mice infected with schistosomula previously exposed to Smcarm1-, GFP-dsRNA, or untreated, from the three biological replicates.
Computer images (Image Pro Plus, Media Cybernetics), from male and female worms captured by a camera (640/480 pixels, RGB) coupled to a light microscope (BX50, Olympus), were used for morphometric analyses. We evaluated the number and area of testicular lobes, ovary area, presence of tubercles, presence of eggs and vitelline glands, and integrity of the tegument (Neves et al., 2004 (link)).
Whole mounts of male and female worms were also analyzed under confocal laser scanning microscopy (LSM-410, Zeiss) using a 543 nm laser and a BP560-615 IR filter, in reflected mode. We examined male (testicular lobes, seminal vesicle) and female (yolk glands, ovary, uterus, and ootype) reproductive systems, as well as the integrity of the tegument and the shape of the oral and ventral suckers.