Figure 4

VHHs 1.2, 1.69, 2.96 and 2.3 do not colocalize with JUNV N- or GPC-specific Mabs in fixed non-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 first incubated with 10 µM of VHHs followed by incubation with a 1:600 dilution of GPC- (a) or N-specific mAbs (b) in a CO₂ incubator. Afterwards the cells were fixed with 2% PFA (without a permeabilization step) and further incubated with an anti-His antibody followed by incubation with the respective secondary antibodies. Anti-His antibody without the presence of VHH was used (−VHH), incubation with a VHH against Flu (VHH Flu) and mock infected cells were used as negative controls. (c) 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.