The amount of condensed tannins in PANE was? ?95% as measured by the acidified vanillin method previously described [22]. extracted three times with 80% acetone (1:10?w/v) and filtrated. The filtrate was evaporated to remove acetone, partitioned with n-hexane and ethyl-ether to remove lipids and then freeze-dried. The yield of the PANE extraction was 16%. The amount of condensed tannins in PANE was? ?95% as measured by the acidified vanillin method previously described [22]. The level Cilazapril monohydrate of endotoxin in the PANE was below the detection limit (0.05 endotoxin unit/mL) using an assay kit (Kinetic-QCL?; Lonza Walkersville Inc., Walkersville, MD). Protocol of animal experiments Male BALB/c mice, 5C6?weeks of age, were purchased from the Animal Breeding Center of the National Taiwan University Hospital (Taipei, Taiwan). The mice were randomized, transferred to plastic cages containing a saw-dust bedding (4C5 mice per cage) and housed in a temperature (23??2C), humidity (60??20%) and light (12-h light/dark cycle)-controlled environment. The present study employed a murine model of food allergy previously described [23]. In brief, mice were randomly divided into the following groups: na?ve (NA), nonsensitized (NS), OVA-sensitized and challenged (OVA), and OVA-sensitized and challenged and PANE-treated (PANE). Mice received PANE via drinking water containing 0.05 and 0.1% (w/v) throughout the entire treatment period. The dosing regimen was chosen according to previous reports showing the immunomodulatory activity of apple polyphenols [9] and the anti-inflammatory, hepatoprotective and immunomodulatory activities of areca Rabbit Polyclonal to OR2T10 nut extracts [14-16,24]. Except for the NA and NS groups, mice were sensitized with OVA by intraperitoneal injection using 0.1?mL sensitization solution containing 50?g OVA and 1?mg alum on day 3 and boosted with a double dose on day 17. Serum of mice was collected prior to OVA challenge on day 31. To induce allergic responses, mice were repeatedly challenged with OVA (50?mg/0.3?mL in saline/mouse) by gavage every other day from day 31 to day 49. Allergic diarrhea characterized as profuse liquid stool was monitored visually for 3?h after challenge. All mice were euthanized 3?h after the last OVA challenge and the spleen and duodenum were harvested for further experimentation. The animal experiments were approved by the Institutional Animal Care and Use Committee of the National Taiwan University. Spleen index The spleen of each mouse was dissected out and weighed immediately after sacrifice. The spleen index was calculated as the spleen weight (mg) per body weight (g). Cellularity of splenocytes Splenocytes were stained with rat anti-mouse CD4 and Gr-1 conjugated with FITC, and rat anti-mouse CD8, CD11b, B220 conjugated with PE-Cy5 antibodies (BioLegend, San Diego, CA) in PBS containing 2% FBS. After washing, the single cell fluorescence of 10,000 cells for each sample was measured by a flow cytometer (BD FACSCalibur, San Jose, CA). Data were analyzed using the software Flowjo 5.7. Histological examination of duodenum The duodenum was excised and fixed in 10% neutral buffered formalin for 2?days. Tissue were embedded in paraffin, sectioned at a thickness of 4C5?m and stained with hematoxylin and eosin (H&E) for routine histopathology. The ratio of villi length over crypt depth was measured by Image-Pro Plus 5.1 morphometric analysis software (Media Cybernetics, Inc. Rockville, MD). Tissue sections were also stained with toluidine blue for identification of mast cells. The number of total and degranulated mast cells was counted manually. Enzyme-linked immunosorbent assay (ELISA) for antibody measurement The levels of total and OVA-specific IgE in serum samples were measured by ELISA [25]. Immunohistochemical staining Tissue sections were deparaffinized and then rehydrated following a standard procedure. The rehydrated slides were immersed in Trilogy? (Cell Marque, Hot Springs, AR) at 121C for 15?min for antigen retrieval. The endogenous peroxidase activity was then quenched with 3% H2O2 in methanol and blocked with normal goat serum. Primary antibodies were applied onto each section overnight. The Cilazapril monohydrate slides were treated with super enhancer, and then incubated with poly-HRP reagent. For visualization, the slides were treated with Cilazapril monohydrate the peroxidase substrate 3-amino-9-ethylcarbazole (AEC) for 2?min followed by hematoxylin counter staining (blue color). For IHC double staining, AEC-treated slides were incubated with another primary antibody at 4C in the dark overnight followed by incubation with AP-conjugated secondary antibody for 1?h. The slides were then treated with AP substrate, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT), for 30?min for observation of a second staining without counter-staining. The number of IHC-positive signals was quantified using the Image Pro Plus 5.1 program. The number of double positive cells showing dark blue surrounded with red color was counted manually. Six duodenums per group were analyzed at 200-fold magnification. Statistical analysis Data of diarrhea occurrence were expressed as percentage and analyzed by the Chi-square test to compare the difference.