Becker K-F, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nährig J, Becker I, Höfler H
Technische Universität, Institut für Pathologie, München, Germany.
J Pathol. 2007 Feb;211(3):370-8. doi: 10.1002/path.2107.
Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.
由于其交联作用，目前认为临床常规处理组织的福尔马林固定会妨碍蛋白质提取和分析。我们研究的目的是开发一种强大、快速、标准化且易于使用的技术，用于从福尔马林固定组织中溶解未降解的全长免疫反应性蛋白质，以进行蛋白质印迹和蛋白质微阵列分析。对各种来源的常规处理福尔马林固定石蜡包埋组织切片进行分析。脱石蜡后，将组织从载玻片上手动切割下来，转移到优化的蛋白质提取缓冲液系统中。溶解蛋白质，随后通过蛋白质印迹和反相蛋白质微阵列进行分析。我们成功地从常规处理的福尔马林固定组织中分离出未降解、可溶且具有免疫反应性的蛋白质。我们能够在预期分子量下检测到膜蛋白、细胞质蛋白和核蛋白。新鲜冷冻组织和福尔马林固定组织之间在蛋白质产量和蛋白质丰度方面未发现差异。使用蛋白质印迹和反相蛋白质微阵列，与免疫组织化学和荧光原位杂交测量相比，在福尔马林固定的乳腺癌活检样本中可靠地测量了受体酪氨酸激酶HER2，这是基于抗体的癌症治疗的重要蛋白质靶点；值得注意的是，免疫组织化学结果不明确的病例（评分2+）可以根据HER2蛋白丰度进行分类。我们新的以临床为导向的多重蛋白质测量系统可能普遍适用于确定少量常规处理的福尔马林固定组织样本中已知疾病相关蛋白质的相对丰度，用于研究和诊断。该技术还可用于识别、表征和验证各种人类疾病中的已知和新蛋白质标志物。
