60 1.4 numerical aperture oil immersion objective

HUVEC and Vero 81 cells cultured on 14 mm coverslips in 24-well plates were infected with R. hoogstraalii str CS and R. rhipicephalii str EH and then incubated at 32°C for 2h. Fresh medium containing 0.5% FBS was added to each well. The infections were allowed to progress for 48h or 96h, and fixed with 3.7% paraformaldehyde for 15 min. The cells were permeabilized with 0.1% Triton X-100 diluted with PBS for 5 min and immersed in 5% bovine serum albumin in PBS for 1h to allow binding with nonspecific sites. The rickettsiae were labeled with R. hoogstraalii str CS or R. rhipicephalii str EH polyclonal antibody (prepared by intraperitoneal injection of each isolated Rickettsia into BALB/c mice) and then with anti-mouse immunoglobulin-Alexa Fluor 488 secondary antibody (Invitrogen) for 30 min at 37°C. F-actin was labeled with Alexa Fluor 568-phalloidin (Invitrogen) for 45 min. All steps were followed by three washes in PBS. Coverslips were sealed with an antifade mounting medium (Invitrogen). Images were acquired on an Olympus microscope with a 60 1.4-numerical-aperture (NA) oil immersion objective (Olympus).

HUVEC and Vero 81 cells cultured on 14 mm coverslips in 24-well plates were infected with R. hoogstraalii str CS and R. rhipicephalii str EH and then incubated at 32°C for 2h. Fresh medium containing 0.5% FBS was added to each well. The infections were allowed to progress for 48h or 96h, and fixed with 3.7% paraformaldehyde for 15 min. The cells were permeabilized with 0.1% Triton X-100 diluted with PBS for 5 min and immersed in 5% bovine serum albumin in PBS for 1h to allow binding with nonspecific sites. The rickettsiae were labeled with R. hoogstraalii str CS or R. rhipicephalii str EH polyclonal antibody (prepared by intraperitoneal injection of each isolated Rickettsia into BALB/c mice) and then with anti-mouse immunoglobulin-Alexa Fluor 488 secondary antibody (Invitrogen) for 30 min at 37°C. F-actin was labeled with Alexa Fluor 568-phalloidin (Invitrogen) for 45 min. All steps were followed by three washes in PBS. Coverslips were sealed with an antifade mounting medium (Invitrogen). Images were acquired on an Olympus microscope with a 60 1.4-numerical-aperture (NA) oil immersion objective (Olympus).