2.7. Indirect Immunofluorescence Assay (IFA) for Detection of Antibodies to EFV

EFV-infected HFK cells were detached by trypsin-EDTA solution and washed three times with phosphate-buffered saline (PBS, pH 7.4) by centrifugation at 200× g for 5 min. The cells were re-suspended in a small volume in PBS and smeared on a 15-well multitest slide glass (MP Biomedicals, LLC, Solon, OH, USA) and then fixed in 100% acetone on ice for 30 min. Equine sera were diluted serially from 1:20 to 320 and incubated with the fixed cells at 37 °C for 30 min in an incubation chamber. The cells were washed three times with PBS. After drying the cells at room temperature, the cells were incubated with 1:80 diluted fluorescein isothiocyanate conjugated goat-anti horse IgG (Jackson Immuno Research Inc., West Grove, PA, USA) at 37 °C for 30 min. After a final wash, infected cells were visualized under a fluorescence microscope (Olympus, Tokyo, Japan). An IFA titer of 20 or greater was regarded as positive. Uninfected HFK cells were used as control cells.