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Negative control staining for IHC were completed by two methods: (1) primary antibody was removed; and (2) primary antibody was replaced by normal species matched sera to the primary antibody. After washing for 30?min with in 0.1?M PBST, the sections were incubated for 2?h with second antibody (Vector Laboratories, Burlingame, CA) diluted 1:500 in PBST at RT. They were washed for 30?min with PBST and placed for 2?h in avidin-biotin-peroxidase complex (ABC kit, Vector Laboratories) diluted 1:1000 in PBST at RT. The immunoreaction were rendered visible by reaction with 0.05?M Tris�CHCl buffer (pH 7.6) containing 0.01% 3,3��-diaminobenzidine (DAB) (Sigma�CAldrich, DAPT St Louis, MO), and 0.0003% H2O2 for 30?min at RT. Finally, slides were counterstained with hematoxylin QS (Vector Laboratories), dehydrated and mounted on glass slides. Eight different areas were randomly chosen from each BML-190 slide under a light microscope with the 400X objective. Digital images were made of each area. To analyze the TGF-��1 and CTGF expression, the percentage of positive cells was measured in each image. For the analysis of Col1 and Col3, cellSens Image Analysis Software (Olympus, Tokyo, Japan) was used to quantify the positive staining areas. Human tendon and a 21-day post-operative canine skin wound were used as positive controls for Col1 and Col3, respectively. Positive control slides were stained and 8 digital images were randomly taken by the same procedures. Observers identified the threshold of the positive area from the digital images of the positive controls on the image software. By using the identified threshold from positive controls, the positive staining areas in both CTS patients and controls were digitally identified. Intraclass correlation coefficients (ICCs) were assessed for the intra and inter-observer reliability of this analysis. Two observers performed the same procedures and measured the positive areas from 30 images which were selected from CTS patients and controls. The measurement Doxorubicin manufacturer of ICCs was performed twice in varied order and separated by at least 1-week. Results were expressed as a mean?��?standard deviation (SD). To assess normality, the Shapiro�CWilk test was used. The Mann�CWhitney test was compared the percentage of TGF-��1 and CTGF positive cells, and the positive area of Col1 and Col3 of CTS patients. Spearman's rank correlation coefficient was used for analysis of correlation between TGF-��1, CTGF, Col1, Col3, and severity of the CTS. Values of p?
