Cytomation liquid dab substrate chromogen system

Sections were deparaffinized through a series of xylene, graded ethanol, and water immersion steps. After autoclaving the sections in target retrieval solution (Dako, Carpinteria, CA, USA; for 15 min, they were incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. Specimens were then incubated overnight at 4° C with anti-ΔNp63 antibody (clone 11F12.1, 1:500 dilution; Millipore, Bedford, MA, USA). After three washes with TBS, the sections were treated with streptavidin-biotin complex (Envision System labeled polymer, HRP, Dako, Carpinteria, CA) for 60 min at room temperature. Immunoreactions were visualized using 3,3′-diaminobenzidine (DAB) substrate-chromogen solution (Cytomation Liquid DAB Substrate Chromogen System, Dako) and counterstained with hematoxylin. Sections were then immersed in an ethanol and xylene bath and mounted on slides for examination. For immunohistochemical analysis, 40 lung SCC tissue samples, 40 pulmonary adenocarcinoma tissue samples and 40 esophageal SCC samples in tissue sections were obtained from patients who underwent surgical treatment at Kawasaki Hospital, Okayama, Japan. The experimental protocol was approved by the Ethics Review Committee of Kawasaki Medical School (Ethics Committee reference number: 1310).

Cores of 2 mm diameter were collected from selected areas of formalin-fixed, paraffin-embedded tissue blocks prepared from 20 normal colon samples and 23 colorectal cancers of adult patients and inserted into tissue microarray (TMA) recipient blocks. Furthermore, 14 histologically intact colonic biopsy samples from children were also examined on protein level. 5 μm thick tissue sections were cut from TMA blocks and from biopsy samples, and were immunostained. Endogenous peroxidase blocking (0.5% hydrogen peroxide and methanol mixture, 30 min, room temperature) and antigen retrieval (Target Retrieval Solution, Dako, Glostrup, Denmark, code: S1699, in pH 6 buffer, performed in a microwave at 900 W for 10 min and at 370 W for 40 min) were carried out on dewaxed samples. Non-specific blocking with 1% bovine serum albumin was applied. Immunohistochemical detection of somatostatin was carried out in a humidified chamber using rabbit anti-human polyclonal antibody (1:50 dilution, overnight, Thermo Scientific, California, USA, code: RB-038-A). EnVision + HRP system (Labeled Polymer Anti-Rabbit, 40 min, Dako, code: K4003) and diaminobenzidine—hydrogen peroxidase—chromogen—substrate system (Cytomation Liquid DAB + Substrate Chromogen System, 10 min, Dako, code: K3468) were used for signal conversion. Finally, hematoxylin co-staining was carried out.