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BACKGROUND Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ would facilitate discovery and validation of molecular pathways underlying neurodegenerative diseases. Immunostaining methods offer in situ detection of targets. Most proposed protocols for multiplexing immunostaining require sophisticated equipment not available for standard labs. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Antibody stripping techniques have not been fully validated for neurodegenerative disease research. Moreover, despite the increased complexity of multiplex immunostaining protocols, quality control steps have not been regularly implemented. 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. RESULTS The proposed protocol using β-mercaptoethanol enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue and antigen integrity. COMPARISON WITH EXISTING METHODS Our proposed method accounts for the intricacies of suboptimal human post-mortem 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 in optimization and validation phases of experiments is prudent. |
