维生素D受体免疫组织荧光染色方法建立

Method establishment of vitamin D receptor immunohistochemical staining

建立维生素D受体(Vitamin D Receptor,VDR)特异性免疫荧光染色方法,对人十二指肠组织中VDR进行测定。采用石蜡包埋的方法对组织进行永久性固定、并制作组织石蜡切片,利用苏木精-伊红(Hematoxylin&Eosin,H&E)方法鉴定组织形态结构的完整性,利用免疫荧光(Immunofluorescence,IF)染色方法对组织中VDR进行免疫荧光染色,确定抗体使用最佳浓度范围并优化实验条件。结果表明:人十二指肠VDR在绒毛上皮细胞的胞质中高表达,一抗D-6的最佳稀释比率为1∶100,二抗A594的最佳稀释比率为1∶250,抗原最佳复性条件为0.01 mol/L p H 6.0柠檬酸缓冲液中850 W功率微波煮沸20 min。按照此方法制作的石蜡切片组织形态结构完整,VDR染色特异性高,重复性好。

A highly specific and sensitive vitamin D receptor（ VDR） immunohistochemical staining was established and was successfully applied to determine VDR cellular expression in human duodenal tissue using paraffin-embedded tissue sections. The HE staining was used to identify the integrity of the tissue structure while the immunofluorescence（ IF） was used for the in situ determination of VDR in the tissue. The experimental conditions were optimized as the following： the dilution for the primary VDR antibody（ D-6） was 1 ∶100,the dilution for the second antibody（ A594） was 1 ∶ 250,the antigen retrieval procedure included： boiling the tissue sections in 0. 01 mol / L p H 6. 0 citrate buffer with 850 W microwave for 20 min and cooling down at the room temperature for 3 h. Using this IF method,VDR was selectively found in human duodenal epithelial cells,indicating high specificity and sensitivity of the method.