Pan Jie, Thoeni Cornelia, Muise Aleixo, Yeger Herman, Cutz Ernest
Division of Pathology, The Hospital for Sick Children, Toronto, ON, Canada.
Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, ON, Canada.
Mod Pathol. 2016 Jun;29(6):557-69. doi: 10.1038/modpathol.2016.52. Epub 2016 Mar 4.
We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine diagnostics.
我们报告了在同一福尔马林固定石蜡包埋（FFPE）切片上进行多标记免疫荧光（MIF）和抗原表位再检测的新方法。MIF方法包括抗原修复步骤，随后进行多标记免疫染色并通过共聚焦显微镜检查。作为示例，我们展示了正常小肠以及先天性肠病（微绒毛包涵体病和先天性簇状肠病）病例中，定位于顶端和基底外侧膜以及肠上皮细胞胞质的表位。我们还展示了胆盐排泄泵蛋白（BSEP）在正常肝细胞胆小管膜以及原发性硬化性胆管炎病例中的定位。为了证明胞质和核表位的共定位，我们分析了正常对照以及增生性肺神经内分泌细胞（PNEC）和神经上皮小体（NEB），它们被认为是支气管肺发育不良（BPD）婴儿肺部的气道传感器。作为胞质标记物，我们分别使用了抗蛙皮素或抗突触小泡蛋白2（SV2）抗体，对于核定位，使用了针对神经源性基因哺乳动物无翅型基因（Mash1）和prospero同源盒蛋白1（Prox1）的抗体，它们对NEB细胞的分化和成熟至关重要，缺氧诱导因子1α（HIF1α）是缺氧反应的下游调节因子以及增殖标记物Ki67。再检测方法包括去除先前免疫标记的靶标并用不同抗体进行免疫染色，便于在PNEC/NEB中的肠上皮细胞刷状缘表位以及HIF1α、Mash1和Prox1的共定位。由于这些方法适用于来自各种组织的常规FFPE病理样本，能够在同一细胞/切片中以优异的对比度和分辨率可视化多个表位，它们适用于诊断病理学中的广泛应用，并且可能特别适用于精准医学诊断。
