Moore Alison M, Boudreau Lee R, Virk Shakeel, LeBrun David P
Department of Pathology and Molecular Medicine, Queen's University; Division of Cancer Biology and Genetics, Queen's Cancer Research Institute.
Queen's Laboratory for Molecular Pathology, Department of Pathology and Molecular Medicine, Queen's University.
J Vis Exp. 2019 Jan 7(143). doi: 10.3791/58735.
Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.
在临床和研究应用中，对福尔马林固定、石蜡包埋（FFPE）组织样本中的目标蛋白质进行定量分析非常重要。一种理想的定量方法应具备准确性、较宽的线性动态范围，并能保持样本的结构完整性，以便识别单个细胞类型。目前的方法，如免疫组织化学（IHC）、质谱分析和免疫印迹，由于其分类性质或需要对样本进行匀浆处理，均无法满足这些要求。作为一种替代方法，我们建议使用免疫荧光（IF）和图像分析来确定FFPE组织中目标蛋白质的相对丰度。在此，我们证明该方法易于优化，具有较宽的动态范围，与定量免疫印迹的金标准相比具有线性可量化性。此外，该方法能够保持样本的结构完整性，并区分各种细胞类型，这在诊断应用中可能至关重要。总体而言，这是一种用于FFPE样本中蛋白质相对定量的可靠方法，并且可以很容易地进行调整以满足临床或研究需求。
