Whole worms of different T. spiralis stages (ML, IIL, AW, and NBL) were fixed with 4% formaldehyde for 30 min and re-fixed with cold acetone for 20 min. The fixed worms were embedded in paraffin and cut into a 2-μm-thick cross-section with a microtome. The expression and tissue location of native TsGS in various worm stages were investigated by indirect immunofluorescent assay (IIFA) (Liu et al., 2017 (link); Cui et al., 2019 (link)). Whole parasites and their cross-sections were blocked with 5% normal goat serum for 2 h and then probed at 37°C for 2 h with 1:10 diluted diverse sera (anti-rTsGS serum, infection serum, and normal serum). After washes with PBS, the worms were stained using goat anti-mouse IgG-FITC conjugate (1:100; Santa Cruz, United States). Following washes again, whole worms and cross-sections were examined under a fluorescence microscope (Olympus, Tokyo, Japan) (Lei et al., 2020 (link); Yue et al., 2020 (link)).
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