Spot digital camera

After 14–18 days, cells were fixed in 4% paraformaldehyde for 1 h at 4 °C. All reagents were diluted in 100 mM PB, pH 7.3. Cells were incubated in blocking solution (0.5% Triton X-100, 3% normal goat serum) for 30 min at room temperature. Two different primary antibodies were simultaneously added to the cells in fresh blocking solution and incubated for 30 min at room temperature. After washing in PB, secondary antibodies were added to the slices in PB containing 0.1% Triton X-100 for 30 min at room temperature. For the analysis of vector expression in Purkinje cells, mouse anti-Calbindin D-28 K (Swant, Marly, Switzerland 1:1000) and polyclonal rabbit anti-GFP (Abcam, Cambridge, UK 1:1000) were used as primary antibodies and goat anti-mouse Alexa 568 (Molecular Probes, Eugene, OR, 1:1000) and goat anti-rabbit Alexa 488 (Molecular Probes, Eugene, OR, 1:1000) were used as secondary antibodies to visualize Purkinje cells [17 (link)]. Stained cells were viewed on an Olympus AX-70 microscope equipped with a Spot digital camera. Recorded images were adjusted for brightness and contrast with Photoshop image processing software. Transfected Purkinje cells were identified by double immunostaining for calbindin and GFP (red and green) and only double positive cells were used for statistical analysis.

Colon specimens were collected, fixed in 10% formalin saline then embedded in paraffin wax. Tissue Sects. (5 μm) were cut and stained with hematoxylin and eosin (H&E). Histopathologic evaluation was done using a light microscope (Olympus America Inc., USA) equipped with a Spot digital camera with a numerical aperture of a high resolution (16-bit digital camera (1280 × 1024) pixels). Scoring was conducted according to Wallace and Keenan [33 (link)]. Briefly, score 0 means intact tissue with no apparent colonic damage; score 1 means that damage is limited to surface epithelium; score 2 means the presence of focal ulceration limited to the mucosa; score 3 denotes the presence of focal, transmural inflammation and ulceration; score 4 denotes the presence of extensive transmural ulceration and inflammation bordered by moderate inflammation of the mucosa, and score 5 denotes the presence of extensive transmural ulceration and inflammation involving the entire section.