TY - JOUR T1 - Multiplex immunofluorescence methods in neurodegenerative diseases JF - bioRxiv DO - 10.1101/533547 SP - 533547 AU - Alexander J. Ehrenberg AU - Dulce Ovando Morales AU - Jorge Santos Tejedor AU - Jan Mulder AU - Lea T. Grinberg Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/05/16/533547.abstract N2 - BACKGROUND The ability to simultaneously localize multiple targets within human tissue sections would markedly facilitate the discovery and validation of molecular pathways underlying neurodegenerative diseases and the understanding of selective vulnerability. The main roadblock to this is successful elution of antibodies or other probes while maintaining tissue and antigen integrity. Although several multiplex techniques involving elution have been proposed, few have been applied or validated in the context of neurodegenerative diseases in human post-mortem tissue. Moreover, despite the increased complexity of these protocols, quality control steps for multiplex techniques have not been regularly or explicitly implemented in studies utilizing them.NEW METHOD Aiming to create protocols for multiplex localization of neurodegenerative processes without the need for specialized equipment, we evaluated several proposed techniques and created a pipeline tailored for the intricacies of human post-mortem brain tissue and suggest quality control steps to ameliorate concerns of cross-reactivity and false positives.RESULTS The proposed protocol using β-mercaptoethanol enables reliable primary and secondary antibody elution 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 examines multiplex immunohistochemistry in the context of neurodegenerative proteinopathies, accounting for the intricacies of human post-mortem tissue. Namely, we account for the need to elute markers of highly aggregated proteins in tissue with significant autofluorescence, diminishing the possible signal-to-noise ratio. Additionally, it explicitly implements and suggests quality control steps to align with the increased complexity associated with multiplex histology.CONCLUSIONS Multiplex immunofluorescence techniques for studying neurodegenerative diseases in postmortem human tissue are feasible and can be implemented in laboratories lacking sophisticated equipment. Nevertheless, the evaluation of elution parameters in the optimization and validation phase of any experiment is prudent. ER -