Immunofluorescence Assay of rHc-MMP-12 Binding to Goat PBMCs

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).