Fitc conjugated goat anti mouse igg secondary antibody

To detect the expression of myc-pBCL-G and HA-pJAB1, SUVEC cells were seeded in 35-mm petri dishes (MatTek, MA, USA) and were co-transfected with pCMV-myc-pBCL-G and pCMV-HA-pJAB1 vectors. About 24 hours after transfection, cells were fixed with 4 % paraformaldehyde for 30 minutes at room temperature and permeabilized with 0.2 % Triton X-100/PBS for 5 minutes. Then, cells were incubated with mouse anti-myc tag (Sigma Aldrich, Saint Louis, USA) or anti-HA tag (Sigma Aldrich, Saint Louis, USA) monoclonal antibody (1:1000 dilution) for 1 hour at room temperature, followed by incubation with FITC-conjugated goat anti-mouse IgG secondary antibody (Sigma Aldrich, Saint Louis, USA, 1:100 dilution) for 1 hour at room temperature. After being rinsed with PBS, cells were observed using an inverted fluorescence microscope.

For complement deposition studies, gonococci, grown as described above, were fixed in 0.5% v/v formaldehyde for 1 h, then resuspended in blocking buffer and finally 10⁵ cells were incubated with 10% v/v NHS (or heat-inactivated NHS) for 30 min at 37°C. For competition experiments, 50 μM of polyP (Graham’s salt, Merck) was added to the reaction mixture. After two washes with 100 μL PBS, the samples were incubated for 1 h with 100 μL FITC-conjugated mouse anti human/mouse C3/C3b/iC3b antibody (1:100 dilution; Caderlane) or mouse anti-human C9 monoclonal antibody (at a 1:250 dilution; Thermo Fisher Scientific). Following washing steps, 100 μL of FITC-conjugated goat anti-mouse IgG secondary antibody was added at a 1:500 dilution and incubated for 30 min (Sigma Aldrich). Finally, cells were resuspended in PBS and bacterial fluorescence was recorded with BD FACS CANTO II (BD Bioscience) and then analyzed using Flow-Jo v.10 (FloJo, LLC).

Mock and CHIKV-infected neurospheres were harvested at the indicated time points. Subsequently, they were fixed, blocked, and frozen for histological sections. The samples were then treated following the same specifications used for phalloidin labeling. Intracellular antibody labeling was performed employing a CHIKV-specific monoclonal antibody (A54Q) (Thermo Fisher Scientific) and a FITC-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich). After incubation, cells were washed extensively with PBS, and slides were mounted with Fluoromount-G^™ mounting medium with DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine), images were captured on a Leica immunofluorescence microscopy DMi8.