Figure 3

VHHs 1.2, 1.69, 2.96 and 2.3 colocalize with JUNV N-specific Mabs in fixed and permeabilized Candid#1 infected cells. Vero cells were mock infected or infected with 150 PFU of Candid#1. At 48 h p.i. cells were fixed, permeabilized and further incubated with 10 µM of VHHs followed by incubation with GPC- (a) or N-specific mAbs (b). Afterwards, monolayers were incubated with an anti-His antibody and finally incubated with the corresponding secondary antibodies. Anti-His antibody without the VHH was included as a negative control (-VHH) and incubation with a VHH against an influenza epitope (VHH Flu) was used as control as well. (c) In addition, immune-staining was performed in mock-infected cells with VHHs followed by incubation with N-specific mAbs. (d) Colocalization of the VHH-specific fluorescent signal with that of the GPC- an N-specific mAbs was determined in 50 cells that showed a positive signal with GPC- or N-specific mAbs. The Pearson’s correlation coefficient (P) was calculated using the Coste’s algorithm provided in the Volocity software package. The scale bar represents 25 µm.