Double immunohistochemical staining method for HIF-1alpha and its regulators PHD2 and PHD3 in formalin-fixed paraffin-embedded tissues.
Double immunohistochemical staining method for HIF-1alpha and its regulators PHD2 and PHD3 in formalin-fixed paraffin-embedded tissues.
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DOI：
10.1097/pai.0b013e3181d6bd59
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发表时间：
2010-07
期刊：
影响因子：
--
通讯作者：
Rustum YM
中科院分区：
文献类型：
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作者：
Vaughan MM;Toth K;Chintala S;Rustum YM
缺氧诱导因子 (HIF-1α) 在缺氧条件下在肿瘤细胞的细胞核中表达，并部分受到细胞质脯氨酰羟化酶 (PHD) 的调节。由于 HIF-1α 在肿瘤细胞中选择性表达，因此正在开发用于癌症治疗的抑制剂。尽管有检测 HIF-1α 和 PHD 的方法，但尚无针对单个肿瘤细胞中这些标记物的免疫组织化学 (IHC) 双染色方法。在方法开发中，使用人鳞状细胞癌 (SCC) 异种移植物 A253 作为无微血管分化良好区域中 HIF-1α 的已知阳性对照组织。该实验室证明这些区域的肿瘤细胞缺氧标志物呈强阳性。另一种人类低分化鳞状细胞癌异种移植物FaDu，没有缺氧区域，被用作阴性对照。使用人肾单独优化 PHD2 和 3 免疫染色。为了优化 HIF-1α 检测，减少了抗原修复的高压锅时间、一抗浓度、扩增试剂和 DAB 显色时间。酪蛋白阻断进一步降低了背景。双重染色导致 HIF-1α (DAB) 细胞核呈棕色，PHD2, 3（坚红）细胞质染色呈粉红色。同种型匹配的对照呈阴性。正常人体组织没有可检测到的 HIF-1α，但表达 PHD2, 3。通过分析 15 个口咽 SCC 手术活检样本（其中 6 个 HIF-1α 呈阳性），证实了这种新的改进方法的潜在实用性。这种新方法定义了检测单个肿瘤细胞中 HIF-1α 和 PHD 的最佳条件，并且具有诊断和治疗潜力。
Hypoxia inducible factor (HIF-1α) expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). Since HIF-1α is selectively expressed in tumor cells, inhibitors are under development for cancer therapy. Although methods for detection of HIF-1α and PHDs are available, an immunohistochemical (IHC) double staining method for these markers in individual tumor cells is not available. For the method development, human squamous cell carcinoma (SCC) xenograft A253 was used as a known positive control tissue for HIF-1α in well differentiated areas without microvessels. This laboratory demonstrated that tumor cells in these areas are strongly positive for hypoxia markers. Another human, poorly differentiated SCC xenograft FaDu, without hypoxic areas, was used as a negative control. PHD2 and 3 immunostaining was optimized individually using human kidney. To optimize HIF-1α detection the pressure cooker time for antigen retrieval, concentration of the primary antibody, amplification reagent, and DAB development time were decreased. Casein blocking further decreased background. The double staining resulted in brown nuclei for HIF-1α (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype matched controls were negative. Normal human tissues had no detectable HIF-1α, but expressed PHD2, 3. Potential utility of this new and improved method was confirmed by analyzing fifteen surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1α. This new method defined optimal conditions for detection of HIF-1α and PHDs in individual tumor cells, and could have diagnostic and therapeutic potential.