In the brain, billions of specialized cells including neurons and microglia together make the complex neuronal connections fundamental to our ability to direct physical, sensory, social and emotional responses. Advancement in the fundamental understanding of the brain, but more broadly in every aspect of scientific progress, comes with the development of new novel analytical techniques and methodologies. In this thesis, we focused our study on developing and applying novel tools to investigate neuronal architecture at the level of protein organization. To summarize, in chapter 2 we first developed a new CRISPR/Cas9 based strategy to label endogenous proteins in neurons. We further expanded on this technique in chapter 3, allowing for duplex labeling and manipulation of multiple endogenous proteins. Second, we described a protocol for single-molecule localization microscopy important for studying protein organization at a sub-micrometer scale (chapter 4), and developed a new analytic method to analyze co-localization in dual-color SMLM datasets (chapter 5). Third, we used the techniques developed in the preceding chapters to study the synaptic organization of AMPAR – auxiliary proteins (chapter 6), and studied the role of the N-terminal domain (NTD) of the AMPAR in synaptic anchoring (chapter 7).