BACKGROUND 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. NEW METHOD 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. RESULTS 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. COMPARISON WITH EXISTING METHODS 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. CONCLUSIONS 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.

中文翻译：

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用于研究神经退行性疾病的手动多重免疫荧光方法。


背景技术神经退行性疾病的特征是蛋白质聚集体的典型沉积物选择性地积聚在脆弱细胞中。同时原位定位多个靶标的能力对于促进致病分子途径的发现和验证至关重要。免疫染色方法可以原位检测特定靶标。有效剥离抗体，允许连续几轮染色，同时保持组织粘附和抗原完整性，是在标准实验室中实现多重免疫染色的主要障碍。此外，剥离技术需要抗体特异性优化、验证和质量控制步骤。新方法为了创建神经退行性相关过程的多重定位协议，而不需要专门的设备，我们评估了几种抗体剥离技术。我们还建议采取质量控制步骤来验证剥离功效，并根据广泛的测试减轻交叉反应和误报的担忧。结果 使用 β-巯基乙醇和 SDS 的方案始终能够在多轮染色中实现可靠的抗体剥离，并最大限度地减少交叉反应的可能性，同时保留人类死后组织中的组织粘附和抗原完整性。与现有方法的比较我们提出的方法对于标准实验室设置来说是最佳的，并且尽管人类死后组织错综复杂并且需要剥离与高度聚集的蛋白质结合的标记，但仍显示出一致的功效。此外，它还结合了质量控制步骤来验证抗体剥离。结论 即使在标准实验室中，用于研究人类死后组织中的神经退行性疾病的多重免疫荧光方法也是可行的。 尽管如此，在实验的优化和验证阶段评估剥离参数是谨慎的。