Fitc conjugated goat anti mouse igg

Detailed procedures for the FFRVN were described previously [36 (link)]. Briefly, pig serum samples were inactivated at 56 °C followed by serial 4-fold dilutions in PBS. 50 μl of each dilution was then mixed with an equal volume of PEDV PC22A-P10 stocks containing 100 fluorescent focus units (FFU). After 1 h incubation at 37 °C, the serum-virus mixtures were inoculated onto confluent Vero cell monolayers. To enhance virus absorption, a brief centrifugation (1000×g for 10 min) was conducted. Subsequently, the inoculum was removed and replaced by 100 μl of DMEM supplemented with 5 μg/ml trypsin. After 4 h incubation, 100 μl of DMEM supplemented with 5% FBS was added to block the action of trypsin and the plates were incubated overnight. The cell culture immunofluorescence assay (CCIF) procedure was then conducted using PEDV N monoclonal Ab (SD6–29) and FITC-conjugated goat anti-mouse IgG (Serotec) as primary and secondary antibodies, respectively. The numbers of FFUs in the wells were counted and the VN Ab titer of each serum sample was expressed as the reciprocal of the dilution giving an 80% reduction in the number of FFU. All sera were tested in triplicate, and the final Ab titer was expressed as the mean of repeats.

IPC colonies were fixed in cold methanol for 15 min at RT, then permeabilized with 0.1% Triton-X100 (Sigma). After that, the background was blocked with 10% donkey serum for 1 h. The primary antibodies, rabbit anti-insulin (Cell signature technology, USA)^{139 (link)} at dilution 1:200 and mouse anti-rat glucagon (Abcam, USA)^{140 (link)} at dilution 1:200, were added and incubated overnight. Then, cyanine (Cy) 3-conjugated donkey anti-rabbit IgG (Bio Legend) and FITC-conjugated goat anti-mouse IgG (BioRad) were used as secondary antibodies, respectively. After incubation with secondary antibodies for 2 h, DAPI was used to stain the nucleus. The results and images were acquired using a fluorescent microscope ZEISS Apotome.2 (Carl Zeiss, Germany) incorporated with Axio Observer Z1 and ZEN pro software (ZEISS International, Germany).