Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Summary

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Abstract

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

Introduction

The understanding and view of the synapse has changed enormously since its first description by Foster and Sherrington in 1897¹. Since then, our knowledge of neuronal communication and the molecular processes behind it has grown exponentially². It has become clear that synapses can be thought of as a two-compartment system: a pre-synaptic compartment containing vesicles for the release of neurotransmitters and a post-synaptic compartment with receptors³. This simplistic view, in the past twenty years, has evolved into a complex network of the proteins required to transduce transmitter binding into....

Protocol

1. Primary cultures

1. Culture mouse hippocampal primary neurons in chambered coverslips (such as Ibidi µ-Slide 8 Well or Nunc Lab-Tek Chambered Coverglass) that match the objective requirement for #1.5 (0.17 mm) coverslip thickness.
2. Coat chambered coverslips with 100 µL of poly-L-lysine (100 µg/mL).
3. The next day, wash the chambered coverslips twice with sterile phosphate-buffered saline (PBS).
4. To obtain mouse primary neurons, isolate hippocampi from P1-P4 pups²³.
5. Place dissected hippocampi in 10 mL of Dissection Media (Table 1) and let them deposit at the bottom o....

Results

We present here the standard workflow to study neuronal proteins co-localization. We first calibrated the microscope and next we performed SIM analysis of the samples. To calibrate the system, we used fluorescent microspheres of 0.1 μm diameter. Upon obtaining super-resolved 3D-SIM images of the beads, the underlying image data are Fourier-transformed to re-convert them to a spatial frequency representation. In Figure 2A, the distinct flower pattern is presented as an indication of super-res.......

Discussion

Elucidating the structure and composition of the synapse is crucial for understanding the physiological and pathological processes that regulate memory and cognition. While in the normal state, synapses are the building blocks of memory, they also underlie complex neurological disorders such as Alzheimer’s disease³². The protocol described here serves to study the co-localization of neuronal proteins with a super-resolution microscopy technique called SIM. Using a particular pattern of illum.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.4% Trypan blue solution	Thermo Fisher Scientific	15250061	Chemical
70 µm filter	Corning	352350	Equiment
Alexa	Thermo Fisher Scientific	-	Antibody
Antibody SENP1	Santa Cruz	sc-271360	Antibody
B27 Supplement	Life Technologies	17504044	Chemical
Bovine serum albumin	Merck	5470	Chemical
CaCl2	Merck Life Science	21115	Chemical
Chambered coverslips	Ibidi	80826	Equiment
DyLight	Thermo Fisher Scientific	-	Antibody
FBS (Hyclone)	GIBCO	SH3007002 (CHA1111L)	Serum
FluoSpheres carboxylate-modified microspheres, 0.1 μm, yellow–green fluorescent	Thermo Fisher Scientific	F8803	Equiment
Glucose	Merck Life Science	G8769	Chemical
Glutamax	GIBCO	35050061	Chemical
HEPES	Merck Life Science	H3537	Chemical
L-Cystein	Merck Life Science	C6852-25g	Chemical
MAP2	Merck	AB15452	Antibody
MEM	Life Technologies	21575022	Medium
MgCl	Merck Life Science	M8266	Chemical
NaOH	VWR International	1,091,371,000	Chemical
Neurobasal A	Life Technologies	10888022	Medium
N-SIM Super Resolution Microscope	Nikon	-	Instrument
Papain	Merck Life Science	P-3125	Chemical
paraformaldehyde	Thermo Fisher Scientific	28908	Chemical
Pen/Strep 10x	Life Technologies	15140122	Chemical
phosphate-buffered saline	Gibco	10010023	Chemical
Poly-L lysine	Sigma	P2636	Chemical
ProLong Diamond Glass Antifade Mountant	Thermo Fisher Scientific	P36970	Chemical
PSD95	NeuroMab	K28/43	Antibody
Round coverglass	Thermo	12052712	Equiment
SUMO1	Abcam	ab32058	Antibody
Synaptophysin	Merck	S5768	Antibody
Triton X-100	Merck	T8787	Chemical
Trypsin inhibitor	Merck Life Science	T9003-500MG	Chemical