<meta charset="utf8"></head><body>Análisis de circuitos sinápticos y localización de proteínas en la retina del ratón

<ol>
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M., Zucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+organization+of+substance+P-like+immunoreactive+amacrine+cells+in+goldfish+retina.">Synaptic organization of substance P-like immunoreactive amacrine cells in goldfish retina.</a> J Comp Neurol. 231 (2), 232-238 (1985).</li><li>Sasso&#232;-Pognetto, M., W&#228;ssle, H., Gr&#252;nert, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycinergic+synapses+in+the+rod+pathway+of+the+rat+retina:+cone+bipolar+cells+express+the+alpha+1+subunit+of+the+glycine+receptor.">Glycinergic synapses in the rod pathway of the rat retina: cone bipolar cells express the alpha 1 subunit of the glycine receptor.</a> J Neurosci. 14 (8), 5131-5146 (1994).</li><li>Hartveit, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+developmental+expression+of+the+NMDA+receptor+subunit+NR2A+in+the+mammalian+retina.">Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina.</a> J Comp Neurol. 348 (4), 570-582 (1994).</li><li>Perkins, G. 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	<tbody>
		<tr>
			<td>1 mL syringe needle</td>
			<td>kangdelai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1% OsO4</td>
			<td>Electron Microscopy Science</td>
			<td>19100</td>
			<td></td>
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			<td>2,2,2-Tribromoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Glutaraldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>16020</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>157-8</td>
			<td></td>
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		<tr>
			<td>Acetone</td>
			<td>Electron Microscopy Science</td>
			<td>10000</td>
			<td></td>
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			<td>Anti-rabbit PKC</td>
			<td>Sigma-Aldrich</td>
			<td>P4334</td>
			<td></td>
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			<td>Anti-Rabbit SP</td>
			<td>Abcam</td>
			<td>ab67006</td>
			<td></td>
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			<td>DAB Substrate kit</td>
			<td>MXB Biotechnologies</td>
			<td>KIT-9701/9702/9703</td>
			<td></td>
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		<tr>
			<td>Elbow scissors</td>
			<td>Suzhou66 vision company</td>
			<td>54010</td>
			<td></td>
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			<td>Electron microscope</td>
			<td>Phillips</td>
			<td>CM120</td>
			<td></td>
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			<td>Epon resin</td>
			<td>Electron Microscopy Science</td>
			<td>14910</td>
			<td></td>
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			<td>forcep</td>
			<td>Suzhou66 vision company</td>
			<td>S101A</td>
			<td></td>
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			<td>Millipore filter paper</td>
			<td>Merck Millipore&#160;</td>
			<td>PR05538</td>
			<td></td>
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		<tr>
			<td>Na2HPO4&#183; 12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>A component of phosphate buffer</td>
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		<tr>
			<td>NaH2PO4&#183; H2O</td>
			<td>Sigma</td>
			<td>71507</td>
			<td>A component of phosphate buffer</td>
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		<tr>
			<td>Picric acid</td>
			<td>Electron Microscopy Science</td>
			<td>19550</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride (NaBH4)&#160;</td>
			<td>Sigma</td>
			<td>215511</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>917R071</td>
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			<td>Ultramicrotome</td>
			<td>Leica</td>
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			<td>Uranyl acetate</td>
			<td>Electron Microscopy Science</td>
			<td>22400</td>
			<td></td>
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			<td>VACTASTAIN ABC kit, Peroxidase (Rabbit IgG)</td>
			<td>Vector Laboratories</td>
			<td>PK-4001</td>
			<td></td>
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investigating retinal circuits and molecular localization by pre&#45;embedding immunoelectron microscopy

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.

<meta charset="utf8"></head><body>Autor destacado: Comprensión de las bases ultraestructurales de la conectividad sináptica de la retina y la localización de neurotransmisores en ratones

Investigación de circuitos retinianos y localización molecular mediante microscopía inmunoelectrónica previa a la inclusión

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is ...

investigating-retinal-circuits-molecular-localization-pre-embedding

Research

JoVE Journal

Neuroscience

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

184 vistas.  Wenzhou Medical University. En nuestra investigación, nos centramos en la fisiología de la retina y la enfermedad. Necesitan investigar la composición celular y los neurotransmisores específicos dentro del circuito neuronal de la retina. El objetivo general es explorar las conexiones sinápticas y la localización celular de los neurotransmisores a lo largo de toda la vía.El mayor desafío de la tecnología de microscopio electrónico inmunológico es difícil lograr un equi...

Video: Autor destacado: Comprensión de las bases ultraestructurales de la conectividad sináptica de la retina y la localización de neurotransmisores en ratones 

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.

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