Experimental infection, parasite isolation, and RNA preparation. A) Immunofluorescence assay using sheep immune serum against T. gondii revealed numerous infected enterocytes. Nuclei are counterstained with DAPI. Shown in the top panel is a 20x magnification of a section of the small intestine (bar = 100 μM) where villi are visible. The bottom panel at 100x magnification shows a schizont containing several merozoites (scale bar = 5 μm). B) Enterocytes containing CZ-strain merozoite stages were stripped away selectively, leaving the villus structure and the cells of the lamina propria intact. Histology section (Hematoxilin & Eosin stained) showing stripped villi at day 5 post infection. C) Microscopic examination of parasites in the detergent washed preparation showed only merozoite stages. D) Quality control of total RNA extracted from parasite preparations separated on an Agilent RNA 6000 Pico Chip. The bands generated by host 28S/18S ribosomal RNA (arrowheads) and parasite 26S/18S ribosomal RNA (arrows) as well as a size marker are indicated. The samples analyzed were: raw, unprocessed material from scraped intestinal lining; Tween 80, material that was syringe-passaged and washed twice with PBS/0.05% Tween 80; Percoll, highly enriched parasite fraction after detergent treatment and Percoll gradient centrifugation; Tachy, RNA prepared from tachyzoites grown in cell culture with human foreskin fibroblasts as host cells.