Alexa fluor 488 tagged phalloidin

Immunofluorescence detection of R. felis in the infected host cells was performed as previously described [83 (link)]. Confluent XTC-2 and Vero cells were infected with an MOI 25. After 72 h, host cells were fixed with 4% paraformaldehyde in cytoskeleton-stabilizing PHEM buffer [84 (link)], permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% milk in PBS. Cells were then incubated with a rabbit polyclonal primary antibody against Rickettsia conorii (1:100; prepared in our experimental animal facility at the Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia). For the localization of intracellular bacteria, we applied a goat anti-rabbit IgG (H + L) polyclonal secondary antibody conjugated with Rhodamine Red-X (Invitrogen, reference number R6394, lot 1402199, made in the USA). To visualize filamentous actin, coverslips were probed with Alexa Fluor 488-tagged phalloidin (Invitrogen, reference number A12379, lot 1378369, made in the United States). Finally, samples were mounted with Vectashield Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI and analyzed using a Zeiss LSM 510 META confocal fluorescent microscope. Brightness and contrast were adjusted by LSM Image browser software (Zeiss, Jena, Germany).