Fitc labelled goat anti rabbit igg

To investigate if CD68 and CD16, CD16 and CX3CR1 or CD16 and CCR2 are co-expressed by cells in situ, double-labelling immunofluorescence stainings were performed. Formalin-fixed paraffin-embedded TAB tissue was deparaffinized and antigen retrieval was performed. Dilutions (1:50) of anti-CD68, anti-CD16, anti-CCR2, or anti-CX3CR1 antibodies were added and incubated overnight. Following washing, FITC-labelled goat anti-rabbit IgG (A11008, Lifetechnologies, Carlsbad, CA, USA) and AF555-labeled donkey anti-mouse IgG (A31570, Lifetechnologies) were used as the secondary antibodies, respectively. DAPI (10236276001, Roche Life Science, Penzberg, Upper Bavaria, Germany) was performed to stain nuclei. Images were taken using Leica DFC345 FX.

To determine binding of murine and rabbit anti-SIC to S. pyogenes cell surface, ~1 × 10⁸ CFU S. pyogenes H584 cells were harvested from overnight culture in THB and washed twice in PBS. Cells were then incubated in a 1 in 10 dilution of serum in PBS from mice immunised with SIC1.300, sham vaccine or (as a positive control) multicomponent Spy7 vaccine candidate^{18 (link)}, or 1 in 100 dilution of purified rabbit anti-SIC antibody (or pre-immune rabbit serum) for 30 min at 37 °C. Cells were then washed twice in PBS-Tween, before incubating with 50 µl of AlexaFluor 647-labelled goat anti-mouse or FITC-labelled goat anti-rabbit IgG (Life Technologies) diluted 1 in 200 in PBS, on ice. Flow cytometry was performed on a FACSCalibur (Becton Dickinson, Oxfordshire, UK) counting 20,000 events. Data were analysed using FlowJo software (Tree Star Inc.).