Anti fade fluoromount solution

The monocytes were separated from freshly collected PBMCs, and washed with PBS (Ca²⁺/Mg²⁺-free, pH 7.4). Cells numbers were adjusted to 1 × 10⁵ cells/ml in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco Life Technologies); then incubated with rFg-CaBP4, or control buffer, at 37 °C in 5% CO₂ for 2 h. Protein binding was examined using an immunofluorescence assay [34 (link)]. The monocytes were washed and added to poly-L-lysine-treated slides, fixed with 4% paraformaldehyde for 30 min and permeabilized with 1% Triton X-100/PBS. The monocytes were incubated with primary rat anti-rFg-CaBP4 IgG antibody (1:200 dilutions), or normal rat serum, overnight at 4 °C, followed by incubation in the secondary goat anti-rat IgG antibody (Beyotime, China) coupled with Cy3 (1:1000 dilutions) for 4 h at 37 °C. Subsequently, nuclei of monocytes were stained with DAPI (Sigma, USA), and protected from the light for 10 min. Anti-fade Fluoromount solution (Beyotime, China) was added to the slides before examination under a Leica TCS CP8 confocal microscope at 100× magnification (Leica Microsystems).

An immunofluorescence assay was performed to evaluate the binding ability of rHc-MMP-12 to goat PBMCs as described previously [30 (link)]. Briefly, freshly collected goat PBMCs were inoculated with 10 μg/ml rHc-MMP-12 and 10 μg/ml purified pET32a tag protein separately in a 24-well plate (1 ml/well). The plate was incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 1 h. Cells were washed in PBS and let to settle on poly-l-lysine coated glass slides for 20 min and fixed with 4% phosphate-buffered paraformaldehyde at room temperature for 30 min. The slides were blocked with PBS containing 5% BSA at 37 °C for 1 h. Subsequently, the slides were incubated with a 1:100 dilution of the primary antibody, rat anti-rHc-MMP-12 sera and normal sera (control slide) for 2 h. After three washes in PBS, slides were incubated in the dark with a secondary antibody, goat anti-rat IgG coupled with Cy3 (Beyotime; 1:1000 dilution) for 30 min. DAPI (Sigma-Aldrich) was subsequently added and the slides were incubated for 5 min at 37 °C in the dark. Finally, after washing slides were covered with a coverslip and immersed in anti-fade fluoromount solution (Beyotime). PBMCs were observed by confocal microscope with laser scanner (PerkinElmer, Waltham, MA, USA) at 100× magnification and digital images were taken using a Nikon microscope software package (Nikon, Tokyo, Japan).

Washed adult worms suspended in PBS were fixed in 4 % formaldehyde-0.2 % glutaraldehyde in PBS for 90 min and then immersed in TISSUE-TeK^® O.C.T. compound (SAKURA, USA). They were snap frozen in liquid nitrogen and stored at -20°C until required for further processing. Cryostat sections of 10 μm thickness were cut, washed with PBS, and treated for 60 min with 10 % normal goat serum in PBS to prevent non-specific binding of antibodies. The sections were then incubated with specific rat anti-rHCcyst-2 antiserum (1:100 dilution) or normal rat serum (control) for 60 min at 37 °C, washed 15 min × 3 with PBS, and subsequently incubated for 60 min with Cy3 goat anti-rat IgG (ab6953, Abcam, Cambridge, MA, USA). Finally, the sections were stained with DAPI (Beyotime, Haimen, Jiangsu, China) to show DNA. After washing with PBS, the specimens were immersed in Anti-Fade Fluoromount solution (Beyotime), which prevents fading of fluorescence during microscopic examination.