Alexa fluor 594 goat anti rabbit igg

To stain intracellular bacteria, cells were permeabilized with PBS containing 0.1% TritonX-100 for 10 min at RT. Cells were then stained against B. henselae following the previously published protocol^{52 (link)}. To stain surface-adhered bacteria, the permeabilization step was omitted and followed by staining against B. henselae.
Imaging was performed using a Nikon Ti2-E motorized fluorescence microscope. Infected and uninfected cells with dye were imaged during the same session with identical acquisition parameters. Fluorescence intensity was optimized on uninfected cells to eliminate the autofluorescence from the cells, and remained constant for all the infected and uninfected cells with the dye.
For differential staining of intracellular and extracellular bacteria, after 48 h, infected DH82 cells were washed and fixed with 4% paraformaldehyde and subsequently incubated with blocking buffer (10% Normal Goat Serum-Gibco, 16-210-072, in PBS) for 1 h. to prevent non-specific binding. After blocking, cells were incubated with the primary antibody: rabbit polyclonal anti–B. henselae (serum derived from a hyperimmunized rabbit eight weeks after inoculation with in vitro propagated B. henselae) at a dilution of 1:300 for 1 h followed by three washes using wash buffer (1X PBS, 0.2% fish skin gelatin). Cells were then incubated with the secondary antibody Goat Anti-rabbit IgG-Alexa Fluor 594 (Thermo Fisher Scientific, R37117) diluted 1:1000 in blocking buffer. After secondary antibody incubation, cells were washed with wash buffer and were permeabilized using 0.1% Triton X-100 in 1X PBS for 20 min. Cells were then incubated with blocking buffer for 1 h and incubated with the primary antibody: rabbit polyclonal anti–B. henselae and counterstained with secondary antibody Goat Anti-rabbit IgG-Alexa Fluor 488 (Thermo Fisher Scientific, A11034) diluted 1:1000 in blocking buffer. Slides were washed and cells were counterstained with DAPI for 10 min to stain nuclei (EMD millipore, 2160), then mounted with the anti-quenching solution (Thermofisher, P36934) and coverslipped.
Imaging was performed using a Nikon Ti2-E motorized fluorescence microscope using a 40X objective and three fluorescence channels (DAPI, FITC, and TRITC) were used to capture an image field. A total of at least 50 images were taken from each individual experiment and yellow dots (extracellular bacteria) were counted manually from each field. The counts from the individual fields were shown (Table S1) and the representative images are shown in (Supplementary Fig. 3)
This experiment was repeated by staining with secondary antibody Goat Anti-rabbit IgG-Alexa Fluor 488 first and then with Goat Anti-rabbit IgG-Alexa Fluor 594. Switching the order of secondary antibodies provided a way to validate specificity and enhance the reliability of our immunofluorescence results. This experiment was repeated two times with secondary antibody combinations. The average percentage of extracellular bacteria from four independent experiments was 12.795%.