FIG. 2.

Double immunofluorescence (IF) of colon histological sections. The sections were dewaxed with xylene and rehydrated through decreasing concentrations of ethanol; tissue antigenicity was recovered using a heat-based retrieval. After unspecific binding sites were blocked with a solution containing 2% swine serum, 2% goat serum, and 1% bovine serum albumin, slides were incubated overnight at 4°C with a mixture of the primary antibodies (anti-B19V VP2 polyclonal rabbit antibody, diluted 1:200, and monoclonal mouse anti-human CD3 clone F7.2.38, diluted 1:40; DakoCytomation) and then in a mixture of fluorescence-conjugated secondary antibodies (fluorescein isothiocyanate polyclonal swine anti-rabbit, diluted 1:200 [DakoCytomation], and rhodamine-conjugated goat affinity-purified antibody to mouse IgG, diluted 1:200 [MP Biomedicals]). Finally, samples were mounted and the nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) using the Prolong antifade reagent kit (Molecular Probes). Negative controls were performed by omitting the primary antibodies. In preliminary experiments, each primary antibody was also tested individually to exclude unspecific cross-reactions with secondary antibodies or optical spillover between green and red signals (data not shown). Microscopy analysis was performed with a DMI6000 B inverted fluorescence microscope (Leica Microsystems SpA, Milan, Italy). Staining with CD3 monoclonal antibody yielded red fluorescence, and staining with anti-B19 VP2 protein (B19p) polyclonal antibody yielded green fluorescence. Cell nuclei were labeled blue with DAPI. Double-stained cells are yellow in the merged image (15). Original IF magnification, ×40. (A) Intraepithelial CD3⁺ T lymphocytes present in a necrotic colonic crypt are infected by B19 virus as shown by expression of viral capsid proteins. Infected CD3 cells are also present in the adjacent stroma. Epithelial cells are detached from the gland basal lamina and show degenerative aspects. (B) On the same tissue section, an adjacent colon crypt shows a normal architecture. Intraepithelial CD3 lymphocytes are not observed, and B19 VP proteins are not expressed. No degenerative aspects are present, and the lamina propria is normal.