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1 材料和方法 1.1 材料 为本院1994/1997手术切除,病理确诊为胃癌72例,其中早期癌17例,中、晚期癌55例,72例痛旁组织中,包括肠上皮化生36例,不典型增生15例,正常上皮21例。鼠抗人Cyclin D1单克隆抗体,兔抗人P16多克隆抗体,相应的生物素化二抗均购于北京中山公司,为美国Zymed公司产品。 1.2 方法 采用免疫组化链霉素亲生物素-辣根过氧化物酶法(SP法)。步骤如下:组织块按常规100mL/L中性福尔马林固定,石蜡包埋,制成厚4μm切片,常规二甲苯脱蜡,梯度酒精水化,PBS洗5min,微波抗原修复,PBS清洗,一抗(工作浓度Cyclin D1为1:20,P16为1:40)室温过夜,PBS洗5min,生物素标记二抗(1:150)15min,PBS洗5min;SP(1:150)15min,PBS洗5min;DAB显色5min～10min,自来水充分冲洗,苏木精复染、脱水、透明、封固,每批实验用已知阳性切片做阳性对照,以
1 Materials and methods 1.1 Materials for the hospital 1994/1997 surgical resection, pathological diagnosis of gastric cancer in 72 cases, of which 17 cases of early cancer, 55 cases of advanced cancer, 72 cases of paranasal tissue, including 36 cases of intestinal metaplasia, 15 cases of dysplasia, 21 cases of normal epithelium. Mouse anti-human Cyclin D1 monoclonal antibody, rabbit anti-human P16 polyclonal antibody, the corresponding biotinylated secondary antibodies were purchased from Beijing Zhongshan company, for the United States Zymed products. 1.2 Methods Immunohistochemical streptomycin biotin - horseradish peroxidase method (SP method). The steps are as follows: The tissue block was fixed with 100 mL / L neutral formalin and embedded in paraffin, sliced at a thickness of 4 μm, deparaffinized by conventional xylene, hydrated with gradient alcohol, washed with PBS for 5 min, washed with PBS, The primary antibody (working concentration Cyclin D1 1:20, P16 1:40) overnight at room temperature, washed with PBS for 5min, biotin-labeled secondary antibody (1: 150) for 15min, washed with PBS for 5min; Wash 5min; DAB color 5min ~ 10min, tap water fully washed, hematoxylin counterstain, dehydrated, transparent, sealed, each batch of positive known positive sections do positive control to