Investigating Retinal Circuits and Molecular Localization by Pre&#45;Embedding Immunoelectron Microscopy

Fenglan Wang; Wenhui Zhong; Jun Zhang

doi:10.3791/66543

JoVE Journal > Neuroscience

신경과학

Investigating Retinal Circuits and Molecular Localization by Pre-Embedding Immunoelectron Microscopy

Published: July 12, 2024

doi:

10.3791/66543

Fenglan Wang*^1,2, Wenhui Zhong*^1,2, Jun Zhang^1,2

¹State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital,Wenzhou Medical University, ²Laboratory of Retinal Physiology and Disease, School of Ophthalmology and Optometry,Wenzhou Medical University

Summary

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.

Abstract

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

Introduction

The complexity of neuronal circuits in the retina presents challenges for exploration, considering the diverse cell types within its structure¹^,². The initial step involves identifying synaptic connections between different cells and determining the cellular localization of specific neurotransmitters or neuropeptides. As molecular biology advances introduce new proteins, precise localization in the retina becomes crucial for understanding their functions and analyzing retinal circuits and synaptic connections³^,⁴^,⁵.

Due to the limited resolution of light microscopy, electron microscopy (EM) is commonly used to detect the subcellular structures of nerve cells. EM has various classifications, with conventional transmission electron microscopy (TEM) utilized for observing cell ultrastructures⁶^,⁷^,⁸^,⁹. Immunoelectron microscopy (immuno-EM), which combines the spatial resolution of EM with the chemical identification ability of antibodies binding specifically to proteins¹⁰, stands out as the optimal and exclusive method for investigating synaptic connections and subcellular protein localization in the retina¹¹^,¹².

Immuno-EM techniques can be divided into pre-embedding and post-embedding methods based on the order of embedding and antibody incubation. Compared with the post-embedding method, the pre-embedding approach is capable of large-scale and long-distance identification¹³^,¹⁴^,¹⁵, offering an optimal approach for studying cell processes like axons and dendrites. Additionally, this technique provides a strong signal and broad field of view, making it advantageous for comprehensive investigations of protein expression and molecular localization in the cytoplasm. This method proves particularly valuable in ensuring chemically identified structures that are visible throughout the entire cytoplasm, cells, or retina.

However, the post-embedding method, while having lower penetration or diffusion compared to the pre-embedding method, is not as sensitive¹⁶^,¹⁷. In simple terms, if the goal is to explore the localization of specific neurotransmitters in the cytoplasm or synaptic terminals, the pre-embedding immuno-EM is the preferred method. Conversely, for identifying the localization of membrane receptors, it is more recommended to utilize post-embedding immunogold EM.

Given these considerations, we opt for the pre-embedding immuno-EM method to delve into retinal circuits, including the interaction between cone and rod pathways, and molecular localization, such as the distribution and synaptic organization of substance P-like immunoreactivity (SP-IR) in the mouse retina.

Protocol

The care and handling of animals were approved by the Regulation of the Ethics Committee of Wenzhou Medical University in accordance with the ARVO guidelines. Adult mice (C57BL/6J, male and female, 8 to 12 weeks of age) were utilized in this research. The equipment and reagents needed for the study are listed in the Table of Materials.

1. Preparation for retina fixation

Assemble the following materials and tools: a dissecting microscope, two forceps with very fine tips, scissors, a 1 mL syringe needle (needle size: G26), and filter paper.
Deeply anesthetize mice by intraperitoneal injection of 2,2,2-tribromoethanol at 0.25 mg/g of body weight. Subsequently, decapitate the mice and cut their heads¹⁶.
Enucleate their eyes with elbow scissors and place them in a glass dish containing 4% paraformaldehyde-0.2% picric acid in 0.1 M phosphate buffer (PB; pH 7.4).
Under the dissecting microscope, punch a hole at the corneal limbus using a 1 mL syringe needle and cut off the anterior segment with scissors, following the hole. Then, remove the lens from the inner retinal surface with forceps.
Use two forceps to carefully peel the sclera until the retina is completely isolated from the eyecup (the cuppy retina tissue separated from the choroid completely). Subsequently, cut the retina into four pieces.
Fix these sections in 4% paraformaldehyde-0.2% picric acid in 0.1 M PB (pH 7.4) for 2 h at room temperature (RT), and then transfer the tissue to 4% paraformaldehyde in 0.1 M PB (pH 10.4) overnight at 4 °C.

2. Pre-embedding immunostaining

After washing in 0.01 M PBS (pH 7.4) six times for 10 min each, incubate the retinal tissues in 1% sodium borohydride (NaBH₄) in 0.01 M PBS (pH 7.4) for 30 min.
Wash the retinal sections in 0.01 M PBS (pH 7.4) at least six times. Meanwhile, remove the vitreous with filter paper and then cut the retina into small slices between 100-300 µm using a double-edged razor blade.
After blocking with 5% normal goat serum (NGS) in 0.1 M PB (pH 7.4) for 1 h at RT, incubate the retinal slices with primary antibodies (Anti-rabbit PKCα, 1:80; Anti-Rabbit SP, 1:100) along with 2% NGS in 0.01 M PBS (pH 7.4) for 2 h at RT on a shaker. Subsequently, incubate for 96 h (5 days) at 4 °C on a shaker.
After washing six times for 10 min each in 0.01 M PBS (pH 7.4), incubate the retinal slices with the secondary antibody at 1:200, such as goat anti-rabbit IgG, along with 2% NGS in 0.01 M PBS (pH 7.4) for 2 h at RT on a shaker. Next, incubate for 48 h (2 days) at 4 °C on a shaker.
NOTE: Secondary antibodies were applied alone in control experiments.
Wash the retinal stripes in 0.01 M PBS (pH 7.4) six times for 10 min each before incubating with reagent A and reagent B from the ABC kit at 1:100 in 0.01M PBS (pH 7.4) for 2 days at 4°C. (Both reagent A and reagent B were diluted with 0.01 M PBS. For example: A solution: 6 μl; B solution: 6 μl; 0.01 M PBS: 588 μl, total of 600 μl).
Wash the retinal stripes in 0.05 M Tris buffer (pH 7.2) three times for 10 min each. Then, pre-incubate the retinal stripes with 5% reagent 1 and 5% reagent 3 from the DAB kit in distilled water for 1 hour at room temperature. (The ratio for example: reagent 1: 50 μl; reagent 3: 50 μl; distilled water: 900 μl, total of 1000 μl).
Stain the retinal stripes with DAB. Add the same volume of solution from three tubes in the DAB kit in sequence (reagent 1-reagent 2-reagent 3). Observe the staining condition using the dissection microscope. Stop the staining when cells become brown; this process takes 10-20 min.
Wash the retinal stripes with 0.05 M Tris buffer (pH 7.2) three times for 10 min each and then wash in 0.01 M PBS six times for 10 min each.

3. Post-fixation and embedding

Fix the retinal stripes in 2% glutaraldehyde for 1-2 h at room temperature (RT), and then wash in 0.01 M PBS (pH 7.4) six times for 10 min each.
Incubate the retinal stripes with 1% osmium tetroxide (OsO₄) in 0.1 M PB for 1 h at RT and keep them in a dark place.
After washing in distilled water (ddH₂O) six times for 10 min each, incubate the retinal tissues with uranyl acetate for 1 h at RT and keep them in a dark place to stain these tissues.
Put the retinal tissues in acetone solutions (50%, 70%, 80%, and 90%) for 10 min each, then in 100% twice for 10 min each.
Dip the retinal tissues in a mixture containing the same volume of uranyl acetate and an epoxy resin for 1 h at 37 °C drying oven, followed by a mixture containing uranyl acetate and the resin (1:4) overnight at 37 °C drying oven.
Transfer the retinal tissues gently using a toothpick into the new resin for 1 h at 45 °C in a drying oven, and then the orientated retinal stripe is embedded in the embedding plate with the resin.
Put the sample in a 45 °C drying oven for 3 h and a 65 °C drying oven for 48 h.
Shape the embedding blocks into trapezoids and cut the block into 1 µm thick sections with an ultramicrotome, stain these sections with toluidine blue, and prescreen regions of interest under a light microscope.
Collect ultrathin sections (70-90 nm) on copper grids and view them under an electron microscope.

Representative Results

Figure 1 shows examples of control experiments without the incubation of primary antibodies against either protein kinase C alpha (PKCα) or SP, in which no immunoreactivity (IR) was found.

Figure 2 depicts the PKCα-IR in the mouse retina. PKCα serves as a marker for all rod bipolar cells (RBC) in the retina¹⁸. At the electron microscopy (EM) level, RBC can be identified through PKCα-IR, visualized by the diaminobenzidine (DAB) reaction product with high electron density. Figure 2A illustrates a PKCα-positive RBC dendrite forming a synapse with a rod terminal in the outer plexiform layer (OPL), while Figure 2B shows a cone-RBC synapse where a PKCα-positive RBC dendrite is post-synaptic to a cone terminal. Additionally, the DAB reaction product aids in identifying RBC terminals (Figure 2C) and axonal processes (Figure 2D) in the inner plexiform layer (IPL).

Figure 3 displays the expression of substance P (SP) in both pre-and post-synaptic connections in the IPL of the mouse retina. SP-IR amacrine cells are presynaptic to SP-negative amacrine cells (Figure 3A) as well as SP-IR amacrine processes (data not shown). Furthermore, SP-IR amacrine cells are post-synaptic to bipolar terminals in the sublayer 3 (S3) and sublayer 5 (S5) levels of IPL, respectively (Figure 3B,C). Our previous research details the analysis of synapses where SP-IR amacrine cell processes form synaptic outputs onto other processes in the IPL¹⁹. Notably, the subcellular localization of SP is mostly found in synaptic vesicles in the presynaptic terminals (Figure 3).

Figure 1: Immuno-electron micrographs from control experiments. Both cone pedicle (CP) with two ribbons (arrowhead) (A) and rod spherule (RS) with one ribbon in the OPL (B) showed no staining. Scale bars: 500 nm (A), 200 nm (B). Please click here to view a larger version of this figure.

Figure 2: Immuno-electron micrographs labeled with PKCα antibody in the mouse retina. (A) RS connects with one PKCα-positive RBC (arrows indicate immunoreactivity) and two horizontal cells (H) in the OPL. (B) CP connects with PKCα-positive RBC in the OPL. (C) and (D) show the PKCα-positive RBC terminal and axonal process in the IPL, respectively. Scale bars: 200 nm (A), 500 nm (B), 500 nm (C), 500 nm (D). Please click here to view a larger version of this figure.

Figure 3: Immuno-electron micrographs labeled with SP antibody in the IPL of mouse retina. (A) SP-IR amacrine cell (A+) is presynaptic to an SP-negative amacrine cell (A-). SP-IR amacrine cells received inputs from bipolar terminals in the S3 (B) and S5 (C) levels of IPL, respectively. Scale bars: 200 nm (A), 200 nm (B), 200 nm (C). Please click here to view a larger version of this figure.

Discussion

This article has described three critical steps for the successful observation of synaptic circuits and protein localization: (1) quick and weak fixation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

We propose that fixation is the key step for a successful pre-embedding immuno-EM approach. Thus, the importance of fresh fixative and fast fixation is emphasized here, naming this principle the "4F principle," which is crucial in tissue preparation. However, achieving both enhanced antibody penetration and effective fixation poses a challenge²⁰. To address this, glutaraldehyde was replaced with picric acid during the initial fixation to improve antibody penetration, as glutaraldehyde can disrupt antigenic determinants and affect immunostaining²¹. Picric acid, in contrast, better preserves membrane structure and tissue integrity²², albeit with a compromise in fixation efficacy. In the third step, 2% glutaraldehyde was used for post-fixation to optimize tissue fixation.

Key points of the protocol presented here include five steps: (1) Early fixation: Utilizing picric acid in the early stage for better antibody penetration while minimizing disruption to antigenic determinants. (2) Post-fixation: Using 2% glutaraldehyde to enhance tissue fixation after immunostaining. (3) Reduction treatment: Incubating tissue in 1% NaBH₄in 0.1 M PB (pH 7.4) for 30 min to restore much of the immunoreactivity and improve the immune reactivity of antigens and antibodies, intensifying positive signals²³. (4) Tissue preparation: Removing vitreous and cutting retinal tissue into small strips to facilitate effective antibody incubation and ensure uniform signals. (5) Moreover, it is imperative to handle the retinal tissue with utmost care, avoiding any damage that could compromise subsequent observations throughout the entire procedure. These measures collectively contribute to the success of the pre-embedding immuno-EM method, allowing one to study diverse aspects such as cone-RBC synapses²⁴, SP-IR molecular localization¹⁹, and the precise expression of α-Syn in the mouse retina²⁵.

Before incubating the first antibody, it is essential to block retinal strips with 2% NGS to eliminate non-specific signal interference. The concentration of the first antibody must be determined based on the antibody itself, typically higher than that used for frozen section staining, and must be optimized beforehand. During the primary antibody incubation period, continuous rotation of the microcentrifuge tube is necessary to ensure full contact of the stacked retinal strips with the antibody, preventing incomplete penetration of stacked parts. To amplify the signal, DAB was pre-incubated before standard DAB staining, with the specific staining time typically ranging from 10-20 min. If the time is too short, the signal may be too weak for observation, and if it is too long, it can lead to false positive signals, affecting accurate judgment. Generally, the incubation time of dyes is directly dependent on the surface color of the tissue (brown to dark brown).

While experiments were performed with freezing tissue in liquid nitrogen to enhance the signal, as mentioned previously¹⁸^,²⁶^,²⁷, this method did amplify positive signals but resulted in poor membrane structure of retinal cells (data not shown). This structural degradation made it challenging to accurately identify the cell types. Considering this, the retinal tissue was not frozen repeatedly. To enhance the positive signal, the incubation time of antibodies was appropriately extended. The proposed protocol combines previous advantages, simplifying steps compared to agar embedding. This not only safeguards the antigenic determinants of proteins but also preserves the integrity of cell membranes. The overarching goal is to explore synaptic connections and cellular localization of neurotransmitters throughout the entire pathway. Therefore, the DAB reaction products in the protocol have not undergone silver intensification treatment, making it more suitable for investigating neurotransmitter-specific synaptic connections and pathways²⁸.

Currently, there are limited methods for the chemical identification of pathways or neurotransmitters at the ultrastructural level. One such method is correlated light and electron microscopy (CLEM)²⁹^,³⁰, which, however, is confined to a very small area and lacks the ability to identify connections throughout the entire pathway. The strengths of pre-embedding immuno-EM lie in its robust signal, wide range, and capability for long-distance analytical positioning and tracking¹³^,¹⁴^,¹⁵. However, this method also has its limitations, such as diffuse signals, making them less specific. For the precise localization of membrane receptors, especially those on the membrane, it may be more suitable to use post-embedding immunogold EM. This technique enables the direct counting of gold particles, facilitating the analysis of the number and variability of these membrane receptors¹⁶.

In the future, the integration of pre-embedding immuno-EM with volume EM holds promise as an effective method for the long-term exploration of identified neural circuits, representing the future perspectives for further development of this method. Volume EM focuses on structure reconstruction, relying on morphology for the identification of specific structures, lacking precise chemical identification³¹^,³². By combining pre-embedding immuno-EM with volume EM, specific structures or substances can be identified during three-dimensional reconstruction, providing a more comprehensive and visually intuitive approach to the identification of neural circuits.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported in part by Grants from the National Key Research and Development Program of China (2022YFA1105503), the State Key Laboratory of Neuroscience (SKLN-202103), Zhejiang Natural Science Foundation of China (Y21H120019).

Materials

1 mL syringe needle	kangdelai
1% OsO₄	Electron Microscopy Science	19100
2,2,2-Tribromoethanol	Sigma-Aldrich	T48402
8% Glutaraldehyde	Electron Microscopy Science	16020
8% Paraformaldehyde	Electron Microscopy Science	157-8
Acetone	Electron Microscopy Science	10000
Anti-rabbit PKC	Sigma-Aldrich	P4334
Anti-Rabbit SP	Abcam	ab67006
DAB Substrate kit	MXB Biotechnologies	KIT-9701/9702/9703
Elbow scissors	Suzhou66 vision company	54010
Electron microscope	Phillips	CM120
Epon resin	Electron Microscopy Science	14910
forcep	Suzhou66 vision company	S101A
Millipore filter paper	Merck Millipore	PR05538
Na₂HPO₄· 12H₂O	Sigma	71650	A component of phosphate buffer
NaH₂PO₄· H₂O	Sigma	71507	A component of phosphate buffer
Picric acid	Electron Microscopy Science	19550
Sodium borohydride (NaBH4)	Sigma	215511
Tris	Solarbio	917R071
Ultramicrotome	Leica
Uranyl acetate	Electron Microscopy Science	22400
VACTASTAIN ABC kit, Peroxidase (Rabbit IgG)	Vector Laboratories	PK-4001

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Wang, F., Zhong, W., Zhang, J. Investigating Retinal Circuits and Molecular Localization by Pre-Embedding Immunoelectron Microscopy. J. Vis. Exp. (209), e66543, doi:10.3791/66543 (2024).