Multicolor Immunofluorescence for Pathogen Detection

After antigen retrieval and blocking, the sample was incubated with mouse anti-talin antibody (1:500) and vinculin recombinant rabbit monoclonal antibody (1:500) for 2 h before incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2000) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:2000) for 1 h. The rickettsia infected sample was incubated with rickettsia rabbit antibody (1:2000) for 2 h before incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:2000) for 1 h. The Ebola infected sample was incubated with Ebola rabbit antibody (1:500) for 2 h before incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:2000) for 1 h. A mouse monoclonal IgG (Thermo Fisher) or Rabbit Polyclonal IgG (Thermo Fisher) served as a negative control [29 (link)]. Fluorescent images were taken and analyzed using an Olympus BX51-microscope or using a Nikon A1R MP ECLIPSE Ti Confocal, with NIS-Elements imaging software version 4.50.00 (Nikon, Tokyo, Japan).