Ded to be decalcified with 10 EDTA solution for 1 week. Samples were embedded in paraffin, and sections were all prepared at a thickness of 4 mm. Sections of the nasal tissues, which were coronally at a distance of 5 mm from the nasal vestibule, were 22948146 stained with HE to calculate numbers of eosinophils in subepithelial mucosa of the nasal septum through a light microscope at 6400 magnification. Inflammation scores in the lung were conducted using a reproducible scoring system as previously described [29]. Briefly, scores were set up and ranged from 0 to 3 based on the levels of peribronchial and perivascular inflammation across main bronchus. The values were given as follows: 0 for no inflammation; 1 for occasional cuffing with inflammatory cells; 2 for most bronchi or vessels surrounded by thin layer (1 to 5 cells) of inflammatory cells, and 3 for most bronchi or vessels were surrounded by a thick layer (more than five cells) of inflammatory cells. For quantifying numbers of eosinophils in the nasal mucosa and lung, five tissue sections of per mouse were Chebulagic acid web randomly selected and carefully assessed in a blinded fashion.ELISA for Cytokines IL-4, 1L-10, IFN-c and IL-2 in Lavage FluidCommercially available ELISA kits were used to assess expression of cytokines IL-4, 1L-10, IFN-c and IL-2 in both NALF and BALF, 3-Amino-1-propanesulfonic acid site according to the instructions of the manufactures. The standard curve range for mouse IL-4, 1L-10, IFN-c and IL-2 were 4?00, 30?000, 15?000, and 2?00 pg/ml, respectively.Flow Cytometry Analysis for Regulatory T cells (Tregs)In order to determine whether the immunomodulatory effects depend on Tregs (CD4+CD25+FoxP3+), percentages of Tregs in paratracheal lymph nodes (PTLN) were determined by one step mouse Treg flow staining kit. PTLN cells were collected at the moment that mice were sacrificed after 24 h of the final challenge and then washed in PBS with 1 FBS. Each tube contained 100 ml of approximately 106 prepared cells. Single cell suspensions were stained for surface molecules anti-mouse FITC-conjugated CD4 and PE-conjugated CD25 as usual, and then stained for intracellular PE-Cy5-conjugated Foxp3 or isotype control after freshly fixation and permeabilization. After staining, the cells were washed and resuspended in an appropriate volume of flow cytometry staining buffer, and subsequently analyzed by flow cytometry (BeckmanCoulter, USA) under instructions of the 
given protocol.Alcian Blue and Periodic Acid Schiff (AB-PAS) Staining for the Nasal Mucosa and LungSections of the nasal tissues and the lung were stained with ABPAS to evaluate goblet cell metaplasia in the airway mucosa. Goblet cells were counted as the blue cells stained positive by ABPAS and percentages were calculated from absolute numbers of cells counted around each airway by using a microscope as previously described [30]. For quantifying goblet cell metaplasia, percentages of AB-PAS positive cells in epithelial areas were assayed from five randomly selected tissue sections of per mouse in a blinded fashion.Statistical AnalysisAll statistical analyses were performed with SPSS v.13.0 (SPSS, USA), and diagrams were done with GraphPad Prism v.5.0 (GraphPad Software, USA). One-way analysis of variance (ANOVO) followed by Student-Newman-Keuls test was used for multiple comparisons of data with Gaussion distribution. The Mann-Whitney U test was applied for data with abnormal distribution. P value less than 0.05 was considered statistical significance.ELISA for Serum OVA-s.Ded to be decalcified with 10 EDTA solution for 1 week. Samples were embedded in paraffin, and sections were all prepared at a thickness of 4 mm. Sections of the nasal tissues, which were coronally at a distance of 5 mm from the nasal vestibule, were 22948146 stained with HE to calculate numbers of eosinophils in subepithelial mucosa of the nasal septum through a light microscope at 6400 magnification. Inflammation scores in the lung were conducted using a reproducible scoring system as previously described [29]. Briefly, scores were set up and ranged from 0 to 3 based on the levels of peribronchial and perivascular inflammation across main bronchus. The values were given as follows: 0 for no inflammation; 1 for occasional cuffing with inflammatory cells; 2 for most bronchi or vessels surrounded by thin layer (1 to 5 cells) of inflammatory cells, and 3 for most bronchi or vessels were surrounded by a thick layer (more than five cells) of inflammatory cells. For quantifying numbers of eosinophils in the nasal mucosa and lung, five tissue sections of per mouse were randomly selected and carefully assessed in a blinded fashion.ELISA for Cytokines IL-4, 1L-10, IFN-c and IL-2 in Lavage FluidCommercially available ELISA kits were used to assess expression of cytokines IL-4, 1L-10, IFN-c and IL-2 in both NALF and BALF, according to the instructions of the manufactures. The standard curve range for mouse IL-4, 1L-10, IFN-c and IL-2 were 4?00, 30?000, 15?000, and 2?00 pg/ml, respectively.Flow Cytometry Analysis for Regulatory T cells (Tregs)In order to determine whether the immunomodulatory effects depend on Tregs (CD4+CD25+FoxP3+), percentages of Tregs in paratracheal lymph nodes (PTLN) were determined by one step mouse Treg flow staining kit. PTLN cells were collected at the moment that mice were sacrificed after 24 h of the final challenge and then washed in PBS with 1 FBS. Each tube contained 100 ml of approximately 106 prepared cells. Single cell suspensions were stained for surface molecules anti-mouse FITC-conjugated CD4 and PE-conjugated CD25 as usual, and then stained for intracellular PE-Cy5-conjugated Foxp3 or isotype control after freshly fixation and permeabilization. After staining, the cells were washed and resuspended in an appropriate volume of flow cytometry staining buffer, and subsequently analyzed by flow cytometry (BeckmanCoulter, USA) under instructions of the given protocol.Alcian Blue and Periodic Acid Schiff (AB-PAS) Staining for the Nasal Mucosa and LungSections of the nasal tissues and the lung were stained with ABPAS to evaluate goblet cell metaplasia in the airway mucosa. Goblet cells were counted as the blue cells stained positive by ABPAS and percentages were calculated from absolute numbers of cells counted around each airway by using a microscope as previously described [30]. For quantifying goblet cell metaplasia, percentages of AB-PAS positive cells in epithelial areas were assayed from five randomly selected tissue sections of per mouse in a blinded fashion.Statistical AnalysisAll statistical analyses were performed with SPSS v.13.0 (SPSS, USA), and diagrams were done with GraphPad Prism v.5.0 (GraphPad Software, USA). One-way analysis of variance (ANOVO) followed by Student-Newman-Keuls test was used for multiple comparisons of data with Gaussion distribution. The Mann-Whitney U test was applied for data with abnormal distribution. P value less than 0.05 was considered statistical significance.ELISA for Serum OVA-s.
