Wieckiewicz J A, Jasiński M, Mytar B, Jasińska A, Uracz W
Department of Clinical Immunology and Microbiology, Copernicus Medical Academy, Cracow, Poland.
Arch Immunol Ther Exp (Warsz). 1993;41(2):165-8.
We adopted the nonradioactive method used for blot hybridization for the detection of inducible mRNA for HLA-DR alpha by the in situ hybridization. Unstimulated and interferon gamma stimulated MonoMac6 and U937 human monocytic cell lines were used as target cells. Sulphonation of plasmid pBR322 with HLA-DR alpha cDNA insert (2 x 700 bp, in Pstl restriction site) was performed according to the manufacturer's procedure (SulfoProbe Kit, Sigma). The hybridization signals were detected with mouse monoclonal, anti-sulphonated DNA antibody, followed by immunovisualization with anti-mouse IgG-alkaline phosphatase conjugates. Unstimulated MonoMac6 and U937 cells showed few granular reaction products only in small percentage of cells (1-5%), while in IFN gamma stimulated cells the fine granular immunoenzymatic reaction was observed in the cytoplasm of majority of cells (> 80%). This method seems to be easy and rapid to perform, making it applicable for routine diagnostic purposes in tissue sections and biopsies.
我们采用用于印迹杂交的非放射性方法，通过原位杂交检测HLA - DRα的诱导型mRNA。未刺激的以及经干扰素γ刺激的MonoMac6和U937人单核细胞系用作靶细胞。按照制造商的程序（磺化探针试剂盒，Sigma）对带有HLA - DRα cDNA插入片段（2×700 bp，位于Pstl限制性位点）的质粒pBR322进行磺化。用小鼠单克隆抗磺化DNA抗体检测杂交信号，随后用抗小鼠IgG - 碱性磷酸酶偶联物进行免疫可视化。未刺激的MonoMac6和U937细胞仅在小部分细胞（1 - 5%）中显示出少量颗粒状反应产物，而在经干扰素γ刺激的细胞中，在大多数细胞（> 80%）的细胞质中观察到精细的颗粒状免疫酶反应。该方法似乎易于操作且快速，使其适用于组织切片和活检的常规诊断目的。
