# Protocol for performing 3D-STORM-based nanoscale organization of NMDA receptors in hippocampal brain tissue

## TL;DR

This paper provides a detailed protocol for using 3D-dSTORM to study the nanoscale organization of NMDA receptors in rat hippocampal brain slices.

## Contribution

The novel contribution is a comprehensive protocol combining 3D-dSTORM and fluorescent self-interference for imaging NMDAR nanodomains in 3D.

## Key findings

- Steps for sample preparation and immunohistochemistry for 3D-dSTORM imaging of NMDARs in brain slices are described.
- The protocol enables super-resolution imaging of NMDAR nanodomains in three dimensions.
- The method allows analysis and comparison of NMDA receptor subtype organization in brain slices.

## Abstract

Direct stochastic optical reconstruction microscopy (dSTORM) unveils ionotropic N-methyl-D-aspartate receptor (NMDAR) organization into discrete nanometer-size domains (nanoclusters) within the postsynaptic density (PSD) of glutamatergic synapses, tuning receptor signaling. Here, we present a protocol to perform 3D-dSTORM imaging of the NMDAR in organotypic and acute rat hippocampal brain slices by combining conventional dSTORM with fluorescent self-interference. We describe steps for sample preparation, immunohistochemistry, 3D-dSTORM acquisition, and image analysis to successfully super-resolve NMDAR nanodomains in three dimensions.

For complete details on the use and execution of this protocol, please refer to Kellermayer et al.,1 Bon et al.,2 and Ferreira et al.3

•Steps to prepare hippocampal brain slices for single-molecule imaging•Instructions for acute brain slice preparation from the rat brain after perfusion with PFA•Guidance on imaging settings to super-resolve NMDA receptor nanodomains in 2D and 3D•Analyze and compare the nanoscale organization of NMDA receptor subtypes in brain slices

Steps to prepare hippocampal brain slices for single-molecule imaging

Instructions for acute brain slice preparation from the rat brain after perfusion with PFA

Guidance on imaging settings to super-resolve NMDA receptor nanodomains in 2D and 3D

Analyze and compare the nanoscale organization of NMDA receptor subtypes in brain slices

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Direct stochastic optical reconstruction microscopy (dSTORM) unveils ionotropic N-methyl-D-aspartate receptor (NMDAR) organization into discrete nanometer-size domains (nanoclusters) within the postsynaptic density (PSD) of glutamatergic synapses, tuning receptor signaling. Here, we present a protocol to perform 3D-dSTORM imaging of the NMDAR in organotypic and acute rat hippocampal brain slices by combining conventional dSTORM with fluorescent self-interference. We describe steps for sample preparation, immunohistochemistry, 3D-dSTORM acquisition, and image analysis to successfully super-resolve NMDAR nanodomains in three dimensions.