Donkey anti rabbit af555

Adherent cultures and hydrogels were fixed with 4% paraformaldehyde at room temperature, then permeabilized using 0.1% TritonX-100 and incubated with blocking solution (PBST with 10% goat serum and 3% BSA) for 1 h at room temperature and stained with the appropriate primary antibody in blocking buffer for 1–2 h at room temperature or overnight at 4 °C. The samples were then washed with PBS, incubated with a secondary antibody, washed and stained with DAPI or Hoechst 34580. Primary antibodies used in this study were: DDR1 (5583, Cell Signaling Technology, clone D1G6, 1:100), E-Cadherin (ab1416, Abcam, Clone HECD-1, 1:100), JAG1 (PA5-46970, Invitrogen, 1:100), Notch1 (4380, Cell Signaling Technology, clone D6F11, 1:200), CK18 (4548 Cell Signaling Technology, clone DC10, 1:250), p63 (13109, Cell Signaling Technology, clone D2K8X, 1:250), CK14 (RB-9020-P, Thermo Fisher, 1:200). Secondary antibodies used were: Rat anti-mouse-APC (550874, BD Bioscience, 1:500), Donkey anti-rabbit-AF555 (A-3157, Invitrogen, 1:500), Donkey anti-rabbit-AF555 (A-31572, Invitrogen, 1:500), Donkey anti-goat-AF488 (A-11055, Invitrogen, 1:500). Donkey-anti-mouse-AF555 (A-31570, Invitrogen, 1:500). Dyes and probes used for immunofluorescence: Phalloidin-AF647 (A22287, Life Technologies, 1:400 of 400× stock), DAPI (D1306, Life Technologies, 1 ug/ml), Hoechst 34580 (H21486, Life Technologies, 1 µg/ml).

Mice were anaesthetized and perfused with 4% PFA in PBS. Heads were post-fixed, decalcified, cryoprotected, frozen and sectioned at 12 µm, as described [40 (link)]. IHC was performed as described [40 (link)]. The following antibodies were used: chicken-anti-βGal (Abcam, ab9361), chicken-anti-GFP (Abcam, ab13970), rabbit-anti-Dsred (Clontech, 632496), donkey-anti-rabbit AF555 (Invitrogen, A31572), goat-anti-chicken AF488 (Invitrogen, A11039). Multi-colour ISH was performed as previously described [67 (link)]. For riboprobes that were used, see the electronic supplementary material, table S1. For quantifying the percentage of OSNs at distinct maturation states in figure 5a,b, every 10th section was collected from anterior to posterior and 42 sections were analysed per mouse.

HBCs and trophoblasts were seeded at a density of 1.0x10⁵ cells per well and infected with ZIKV or ZIKV+DENV nAbs at a multiplicity of infection (MOI) of 0.5 for 48 hours. FcγR-blocking antibodies were added to the cells one hour prior to infection at a concentration of 10 μg/mL. In some conditions, protein G (1.5 μg/mL) was added to ZIKV–DENV nAbs immune complexes and incubated for one hour before adding this to the cells. For visualization with confocal laser scanning microscopy (Zeiss), cells were fixed and permeabilized (BD Cytofix/Cytoperm) and stained with primary antibodies against CD68 (DAKO, 1:75 dilution), cytokeratin-7 (Abcam, ab52870, 1:350 dilution) and 4G2 (Merck Millipore, 1:200) and secondary antibodies goat-anti mouse IgG1 A647, goat-anti mouse IgG2A AF488 and donkey anti-rabbit AF555 (all Invitrogen, 1:400 dilution). Nuclei were stained with Hoechst 33342 (Invitrogen). Percentage of infected cells and purity of cells were calculated using ImageJ. Processing of the confocal laser scanning microscopy images was done with the ImageJ plugin QuickFigures [27 ].