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was fixed by high temperature and pressure with citrate buffer solution (Maixin_bio MVS-0066). Each section was added 100 μl 3% H2O2 to block endogenous peroxidase activity in room temperature for 20 minutes. After washing by phosphate buffer solution (PBS, Maixin_bio PBS-0060/0061), 100 μl primary antibody (Santa cruze SC-6014, Notch-1) were incubated at 4°C overnight. And then followed by polink-2 plus Polymer HRP detection system for Goat Primary antibody (ZSGB-BIO, PV-9003), used reagent 1 100 μl each section at room temperature for 20 minutes, later washed off, reagent 2 was the same operation. Afterwards, diaminobenzidine (DAB, biogenex, HK1240411) was used as the color reagent before slides were counterstained with hematoxylin, then dehydrated step by step by using descending concertrations of ethanol, cleared with xylene, mounted with neutral gum. Simultaneously, using Palbociclib clinical trial PBS (0.01 mol/L, PH = 7.4) instead of primary antibody as blank control. All the sides were independently assessed by two pathologists. The immunostained results of Notch-1 protein were semi-quantitated

according to the criteria from published literatures [2, 11]. Each section randomly selected 5 high-power fields, positive cells represented by the percentage of totally number of similar cells. Details were as follows: 0 point for less than 5% positive cells; 1 for 5%-25% positive cells; 2 for 26%-50% positive cells; 3 for 51%-75% positive cells; 4 for more than 76% positive PF-02341066 mw cells. The staining intensity was scored on a scale as weak, moderate or strong. 0 point for no stained; 1 for low Sodium butyrate stained (pale yellow); 2 for moderate stained (brown); 3 for strong stained (tan). After added the two scores, <3 was defined as negative, ≥3 was positive. Statistical analysis The statistical analyses were performed using software SPSS version 17.0 (SPSS Inc, Chicago). Individual clinical information and pathological characteristics were summarized using descriptive statistics. Qualitative data were determined

a possible clear correlation analysis by chi-square test or Fisher’s exact test if the number was less than 5. Survival time was measured from the date of surgery to the latest follow-up or the date of death. Univariate analysis, including Survival analysis, was estimated by Kaplan-Meier method. Log-rank test was used for comparison of survival rate. Cox proportional hazards regression model was used for multivariate analysis. P < 0.05 was considered to demonstrate statistical significance. Results Notch-1 expression in LAD cell lines or tissues First, nuclear acid detection and Western blot assays were performed to detect the expression of Notch-1 in a normal human bronchial epithelial cell line (16HBE) and three human LAD cell lines (SPC-A1, A549 and H1299).