Alexa 555 goat anti rabbit secondary antibody

Briefly, cells were fixed with 4% paraformaldehyde buffered in PBS for 10 min. Subsequently, the cells were permeabilized using PBS containing 0.5% Tween20 for 15 min. To block non-specific interactions, cells were incubated with 5% FBS in PBS for 1 hr. Lysosomes were stained using lysotracker deep red (Thermo Fisher Scientific) prior to fixation. Early endosomes were stained using Alexa 647conjugated anti-EEA1 (Abcam). PLY was stained using mouse anti-PLY (Abcam) and Alexa488-goat anti-mouse secondary antibody (Thermo Fisher Scientific). MRC-1 was detected using rabbit anti-MRC-1 (Abcam) and Alexa 555-goat anti-rabbit secondary antibody (Thermo Fisher Scientific). Pneumococci were stained using rabbit anti-pneumococcal anti-serum (Eurogentec) labeled with Alexa 488 using Zenon rabbit IgG labeling kit (Thermo Fisher Scientific). Type 1 clinical strains were stained using anti-serum Type 1 (Statens Serum Institut). Samples were washed twice with PBS between the antibody incubations and mounted on slides using ProLong Diamond antifade reagent containing DAPI (Thermo Fisher Scientific). Images were acquired using the Delta Vision Elite microscope under the 100x objective (GE Healthcare).