Date of Completion
Linnaea Ostroff, Joseph Loturco, Alexander Jackson
Field of Study
Physiology and Neurobiology
Master of Science
To fully understand how biological systems give rise to complex phenomena, both structural and functional knowledge of the components in the system must be acquired. In this manuscript, I explain why new methods must be developed to achieve this level of understanding with brain tissue and introduce serial multiplexing as a potential solution. I discuss how this method can lead to highly annotated volume reconstructions of brain tissue using electron and light microscopy, along with the steps required to do so. I present what our lab has found throughout the process of adapting, refining, and combining more traditional methods together to work harmoniously for achieving multiplexed labeling of brain tissue. I also discuss what methods we found to be incompatible and what our advice is for others looking to achieve similar results. I explain why electron microscopy is required to extract ultrastructural information of brain tissue such as synapses, dendritic morphology, and subcellular components. Topics including sample preservation and storage, serial sectioning, grid handling, and immunohistochemistry, among others, are also discussed. To perform immunohistochemistry on brain tissue without compromising its structural integrity in the electron microscope is a considerable challenge. This challenge is magnified when the goal is to label an unrestricted number of target proteins in a sample destined for volume reconstruction, especially considering the lack of a unified roadmap for doing so. We therefore conducted experiments to test the efficacy and reliability of established techniques throughout all processes of obtaining brain tissue for serial multiplexed electron microscopy. We also tested the efficacy and dilution requirements of antibodies across several parameters, targeting candidate proteins and molecules of interest using immunofluorescence microscopy. We draw preliminary conclusions regarding our findings and explore possible directions for the project to continue in the future. The work presented in this manuscript reflects the concerted effort of many individuals over a year and a half and was unfortunately forced to stop prematurely due to a public health crisis beyond our control.
Tiley, Peter, "A Serial Multiplexed Approach to Immunolabeling Brain Tissue for Electron Microscopy" (2020). Master's Theses. 1503.