Immunocytochemistry of Intracellular Parasites

Immunocytochemistry was performed on paraformaldehyde-fixed cells as described earlier (Martens et al., 2004 (link); 2005 (link);); the images were taken with an Axioplan II fluorescence microscope and AxioCam MRm camera and processed by Axiovision 4.7 (all Zeiss) and Image J softwares (http://rsb.info.nih.gov/ij/). 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, Invitrogen) was used for nuclear counterstaining at a final concentration of 0.5 µg ml⁻¹. Intracellular parasites were identified by observing the vacuolar localization of the T. gondii protein GRA7 or by distinct pathogen appearance in phase contrast.