Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Summary

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Abstract

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues¹. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback². The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure^3-5.

Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO₄), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO₄ is highly reactive and has been shown to mask antigens⁶, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO₄ with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues⁷. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.

We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously⁸, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function^8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca²⁺/CaM-dependent protein kinase II (CaMKII)¹⁰. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine⁸. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

Protocol

1. Fixation

Fixatives are carcinogenic; wear gloves and handle the fixatives in a fume hood. Unless otherwise noted, all incubations are done on ice and all solutions should be filtered before use. Use electron microscopy-grade reagents.

Day 1

1. After experimental conditions (e.g. viral injection, drug treatment), place the membrane with organotypic hippocampal slices in a 60 x 15 mm polystyrene Petri dish containing ice-cold 0.1 M phosphate buffer (Sorensen's phosphate buffer), pH 7.3. Add 1 - 2 ml of 0.1 M phosphate buffer directly on top of the slices to keep them cold.

Results

Figure 2B shows an example of the distribution of endogenous Ng molecules in dendritic spines of CA1 hippocampal pyramidal neurons. Nickel grids with ultrathin (60 nm) tissues containing CA1 region of the hippocampus (as seen in Figure 2A) were covered in 1% T/PB, 50 mM glycine, then blocked with 2.5% BSA and 2.5% serum prior to incubation with anti-Ng antibody. After washing with T/PB, grids were then covered in anti-rabbit secondary antibody coupled to 10 nm gold. Finally, grids were w.......

Discussion

In this protocol, we have adopted the Phend and Weinberg method for brain and spinal cord tissues to study dendritic spines in rat hippocampal slice cultures. Dendritic spines in the hippocampal CA3-CA1 area are delicate structures containing a vast variety of proteins that play important roles in regulating neuronal functions. The presented method provides a balanced approach for achieving enhanced antigenicity while maintaining good ultrastructural preservation (Figure 2A), permitting reasonable.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
60 x 15 mm polystyrene Petri dish	Falcon	351007
Disposable scalpel	EXELINT	29552
Cell culture inserts	Millipore	PICM03050
10 nm Goat-anti-rabbit gold	Electron Microscopy Sciences	25108
Anti-Neurogranin antibody	Millipore	AB5620
100% Picric acid	Electron Microscopy Sciences	19550
96% Paraformaldehyde	Acros Organics	AC41678-0030
25% Glutaraldehyde (EM grade)	Sigma	G5882
Uranyl acetate	Electron Microscopy Sciences	22400
p-Phenylenediamine	Sigma	P6001
Platinum (IV) chloride	Sigma	379840
Tannic acid	Electron Microscopy Sciences	21710