Fitc labeled goat anti mouse igg

The baby hamster kidney cells (BHK-21) were plated in 6-well tissue culture plates (Nunc) at 2 × 10⁶ cells per well in RPMI-1640 containing 5% FBS [22 (link)]. The recombinant pcDNA3.1-TsNd and pcDNA3.1 (1 × 10⁸/mL) were transfected into the BHK-21 cells with Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s instructions. For RT-PCR assays, total RNA was extracted from the transfected BHK-21 cells using TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. The cDNA samples were obtained using a cDNA First Strand Synthesis Kit (Bioer Technology, China). Transcription of the TsNd gene in the transfected BHK-21 cells was analyzed by RT-PCR using specific primers listed above. For IFT, BHK-21 cells expressing TsNd proteins were cultured as monolayers and washed with PBS and fixed with 4% acetone at room temperature for 20 min. Then, cells were washed with PBS and permeabilized with 1% TritonX- 100 at 4°C for 10 min, after which the cells were further incubated with mouse anti-rTsNd serum (1:10 dilution) at 37°C for 1 h. After washing three times in PBS, the cells were incubated with a 1:50 dilution of FITC-labeled goat anti-mouse IgG (Santa Cruz, USA) in PBS at room temperature for 1 h, after washing three times in PBS, and examined under a fluorescent microscope (Olympus, Japan).

The recognition of the native TsNd in T. spiralis different developmental stages (ML, IIL, AW, and NBL) was observed by IFT as described previously [25 (link),26 (link)]. The intact whole worms were fixed in aceton and further incubated with anti-rTsNd serum from mice immunized by TsNd DNA (1:10 dilution) at 37°C for 1 h. After being washed three times in PBS, the whole parasites were incubated with a 1:50 dilution of FITC-labeled goat anti-mouse IgG (Santa Cruz, USA), washed five times in PBS, and examined under a fluorescent microscope (Olympus, Japan).

IFA was carried out to confirm the expression of TsASP2 at diverse T. spiralis stages. Various T. spiralis worm stages (ML, IIL, AW and NBL) were fixed in paraformaldehyde and embedded in paraffin. The 2-μm-thick sections were prepared using a microtome. After blocking with 5% normal goat serum, the sections were incubated with anti-rTsASP2 serum (1:50 dilutions) at 37°C for 1 h. After washing with PBS, they were incubated with FITC-labeled goat anti-mouse IgG (1:100 dilution, Santa Cruz, USA) and observed under a fluorescence microscope (Olympus, Japan) [41 (link)].