Ehrenberg Alexander J, Morales Dulce Ovando, Piergies Antonia M H, Li Song Hua, Tejedor Jorge Santos, Mladinov Mihovil, Mulder Jan, Grinberg Lea T
University of California, San Francisco, Memory and Aging Center, Weill Institute for Neurosciences; San Francisco, CA, USA; University of California, Berkeley, Helen Wills Neuroscience Institute; Berkeley, CA, USA; University of California, Berkeley, Dept. of Integrative Biology; Berkeley, CA, USA.
University of California, San Francisco, Memory and Aging Center, Weill Institute for Neurosciences; San Francisco, CA, USA.
J Neurosci Methods. 2020 Jun 1;339:108708. doi: 10.1016/j.jneumeth.2020.108708. Epub 2020 Mar 31.
Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps.
Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing.
A protocol using β-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue.
Our proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping.
Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.
神经退行性疾病的特征是蛋白质聚集体的典型沉积，这些聚集体选择性地在易损细胞中积累。同时在原位定位多个靶点的能力对于促进致病分子途径的发现和验证至关重要。免疫染色方法能够在原位检测特定靶点。有效去除抗体，允许连续多轮染色同时保持组织黏附性和抗原完整性，是标准实验室进行多重免疫染色的主要障碍。此外，去除技术需要针对抗体进行优化、验证和质量控制步骤。
为了创建无需专门设备即可对神经退行性相关过程进行多重定位的方案，我们评估了几种抗体去除技术。我们还基于广泛测试推荐了质量控制步骤，以验证去除效果并减轻对交叉反应性和假阳性的担忧。
使用β-巯基乙醇和十二烷基硫酸钠的方案能够在多轮染色中始终实现可靠的抗体去除，将交叉反应性的几率降至最低，同时在人类尸检组织中保持组织黏附性和抗原完整性。
我们提出的方法在标准实验室环境中是最优的，尽管人类尸检组织不太理想且需要去除与高度聚集蛋白结合的标记物，但仍显示出一致的效果。此外，它纳入了质量控制步骤以验证抗体去除效果。
即使在标准实验室中，用于研究人类尸检组织中神经退行性疾病的多重免疫荧光方法也是可行的。然而，在实验的优化和验证阶段谨慎评估去除参数是明智的。
