Abstract
BACKGROUND The ability to routinely visualize multiple targets within human tissue sections would markedly facilitate the discovery and validation of molecular pathways underlying neurodegenerative diseases. Several techniques have been proposed, but few have been thoroughly evaluated in the context of human tissue in neurodegenerative diseases associated with tau protein deposition.
NEW METHOD We reviewed and evaluated several established techniques for multiplex immunohistochemistry and immunofluorescence and created a pipeline tailored for the intricacies of human brain postmortem tissue. We also make recommendations on appropriate use and necessary validation steps for implementing multiplex immunofluorescence methods in the setting of neuropathology laboratories focused on neurodegenerative diseases without the need of sophisticated equipment.
RESULTS The proposed protocol enables reliable primary and secondary antibody elution and minimize the odds of cross reactivity. Out of all tested methods, β-mercaptoethanol reliably eluted all antibodies tested and maintained antigen integrity.
COMPARISON WITH EXISTING METHODS Several techniques for multiplex immunohistochemistry have been widely used in the literature, however few experiments explicitly validate elution parameters in the specific experimental setup. Here, we illustrate the necessity of this validation, as well as examine the longevity of tissue samples and antigens across five rounds of immunostaining.
CONCLUSIONS Multiplex immunofluorescence protocols for studying neurodegenerative conditions in postmortem human studies are feasible and can be implemented in laboratories lacking sophisticated equipment. Nevertheless, the inclusion of elution parameters in the optimization and validation phase of any experiment is prudent. Furthermore, elution control slides should be incorporated into multiplex immunohistology experiments that rely on effective elution of antibodies during any step.