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).

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
<ol>
	<li>Assemble the following materials and tools: a dissecting microscope, two forcep...

Synaptic Circuits Analysis and Protein Localization in the Mouse Retina

<ol>
	<li>Chua, N. J., 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=A+complete+reconstruction+of+the+early+visual+system+of+an+adult+insect.">A complete reconstruction of the early visual system of an adult insect.</a> Curr Biol. 33 (21), 4611-4623.e4614 (2023).</li><li>Ravi Chander, P., Hanson, L., Chundekkad, L., Awatramani, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+underlying+multi-feature+extraction+in+the+retina.">Neural circuits underlying multi-feature extraction in the retina.</a> J Neurosci. , (2023).</li><li>Sirivisoot, S., 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=Development+and+characterization+of+mouse+anti-canine+PD-L1+monoclonal+antibodies+and+their+expression+in+canine+tumors+by+immunohistochemistry+in+vitro.">Development and characterization of mouse anti-canine PD-L1 monoclonal antibodies and their expression in canine tumors by immunohistochemistry in vitro.</a> Vet Q. 43 (1), 1-9 (2023).</li><li>Raeisi, H., 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=Development+and+characterization+of+phage+display-derived+anti-toxin+antibodies+neutralizing+TcdA+and+TcdB+of+Clostridioides+difficile.">Development and characterization of phage display-derived anti-toxin antibodies neutralizing TcdA and TcdB of Clostridioides difficile.</a> Microbiol Spectr. 11 (5), e0531022 (2023).</li><li>Ryschich, A., Dong, Y., Sch&#228;fer, M., Ryschich, E., Karakhanova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DWH24:+A+new+antibody+for+fluorescence-based+cell+death+analysis.">DWH24: A new antibody for fluorescence-based cell death analysis.</a> Methods Appl Fluoresc. 11 (4), (2023).</li><li>Jastrzebska, B., 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=Functional+characterization+of+rhodopsin+monomers+and+dimers+in+detergents.">Functional characterization of rhodopsin monomers and dimers in detergents.</a> J Biol Chem. 279 (52), 54663-54675 (2004).</li><li>Huang, P., 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=Mechanism+of+Shenfu+injection+in+suppressing+inflammation+and+preventing+sepsis-induced+apoptosis+in+murine+cardiomyocytes+based+on+network+pharmacology+and+experimental+validation.">Mechanism of Shenfu injection in suppressing inflammation and preventing sepsis-induced apoptosis in murine cardiomyocytes based on network pharmacology and experimental validation.</a> J Ethnopharmacol. 322, 117599 (2024).</li><li>Maheshwari, U., 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=Inorganic+phosphate+exporter+heterozygosity+in+mice+leads+to+brain+vascular+calcification,+microangiopathy,+and+microgliosis.">Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis.</a> Brain Pathol. 33 (6), e13189 (2023).</li><li>Mackiewicz, J., 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=Effect+of+gravity+in+long-term+vitreous+tamponade:+In+vivo+investigation+using+perfluorocarbon+liquids+and+semi-fluorinated+alkanes.">Effect of gravity in long-term vitreous tamponade: In vivo investigation using perfluorocarbon liquids and semi-fluorinated alkanes.</a> Graefes Arch Clin Exp Ophthalmol. 245 (5), 665-675 (2007).</li><li>Luj&#225;n, R., Rubio, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editorial:+Immunoelectron+microscopy:+Placing+molecular+functions+within+a+neuronal+context.">Editorial: Immunoelectron microscopy: Placing molecular functions within a neuronal context.</a> Front Neuroanat. 16, 1043371 (2022).</li><li>Xu, S., 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=Synaptic+changes+and+the+response+of+microglia+in+a+light-induced+photoreceptor+degeneration+model.">Synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model.</a> Mol Vis. 27, 206-220 (2021).</li><li>Chadha, A., Volland, S., Baliaouri, N. V., Tran, E. M., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+route+of+the+visual+receptor+rhodopsin+along+the+cilium.">The route of the visual receptor rhodopsin along the cilium.</a> J Cell Sci. 132 (10), jcs.229526 (2019).</li><li>Huang, H. J., 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=Multiple+nucleocapsid+structural+forms+of+shrimp+white+spot+syndrome+virus+suggests+a+novel+viral+morphogenetic+pathway.">Multiple nucleocapsid structural forms of shrimp white spot syndrome virus suggests a novel viral morphogenetic pathway.</a> Int J Mol Sci. 24 (8), 7525 (2023).</li><li>Tissarinen, P., 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=Elevated+human+placental+heat+shock+protein+5+is+associated+with+spontaneous+preterm+birth.">Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth.</a> Pediatr Res. 94 (2), 520-529 (2023).</li><li>Huang, Z., 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=A+neural+tract+tracing+study+on+synaptic+connections+for+cortical+glutamatergic+terminals+and+cervical+spinal+calretinin+neurons+in+rats.">A neural tract tracing study on synaptic connections for cortical glutamatergic terminals and cervical spinal calretinin neurons in rats.</a> Front Neural Circuits. 17, 1086873 (2023).</li><li>Zhang, J., Petralia, R. S., Wang, Y. X., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+quantitative+immunogold+analysis+of+membrane+receptors+at+retinal+ribbon+synapses.">High-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses.</a> J Vis Exp. 108, e53547 (2016).</li><li>Ottersen, O. P., Landsend, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+glutamate+receptors+at+the+synapse.">Organization of glutamate receptors at the synapse.</a> Eur J Neurosci. 9 (11), 2219-2224 (1997).</li><li>Greferath, U., Gr&#252;nert, U., W&#228;ssle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod+bipolar+cells+in+the+mammalian+retina+show+protein+kinase+C-like+immunoreactivity.">Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity.</a> J Comp Neurol. 301 (3), 433-442 (1990).</li><li>Wang, F., 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=Distribution+and+synaptic+organization+of+substance+P-like+immunoreactive+neurons+in+the+mouse+retina.">Distribution and synaptic organization of substance P-like immunoreactive neurons in the mouse retina.</a> Brain Struct Funct. 228 (7), 1703-1724 (2023).</li><li>van Opbergen, C. J. M., 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=34;Orphan&amp;#34;+Connexin43+in+Plakophilin-2+deficient+hearts+revealed+by+volume+electron+microscopy.">34;Orphan&amp;#34; Connexin43 in Plakophilin-2 deficient hearts revealed by volume electron microscopy.</a> Front Cell Dev Biol. 10, 843687 (2022).</li><li>Li, S., 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=Safe+and+efficient+oral+allergy+immunotherapy+using+one-pot-prepared+mannan-coated+allergen+nanoparticles.">Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles.</a> Biomaterials. 303, 122381 (2023).</li><li>Yamashita, K., Kusakabe, M., Sano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+of+dissociating+hepatocytes+from+fixed+liver+of+the+mouse.">A simple and rapid method of dissociating hepatocytes from fixed liver of the mouse.</a> Stain Technol. 56 (1), 29-33 (1981).</li><li>Tanabe, T., Shin, M., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoelectron+microscopy+study+of+polyamines+using+a+newly+prepared+monoclonal+antibody+against+spermidine:+use+of+a+mixture+of+glutaraldehyde+and+paraformaldehyde+as+a+cross-linking+agent+in+the+preparation+of+the+antigen.">Immunoelectron microscopy study of polyamines using a newly prepared monoclonal antibody against spermidine: use of a mixture of glutaraldehyde and paraformaldehyde as a cross-linking agent in the preparation of the antigen.</a> J Biochem. 135 (4), 501-507 (2004).</li><li>Xiao, J., 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=Rod+bipolar+cells+receive+cone+photoreceptor+inputs+through+both+invaginating+synapses+and+flat+contacts+in+the+mouse+and+guinea+pig+retinas.">Rod bipolar cells receive cone photoreceptor inputs through both invaginating synapses and flat contacts in the mouse and guinea pig retinas.</a> J Comp Neurol. 531 (11), 1184-1197 (2023).</li><li>Yang, Q., 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=Expression+of+&#945;-Synuclein+in+the+mouse+retina+is+confined+to+inhibitory+presynaptic+elements.">Expression of &#945;-Synuclein in the mouse retina is confined to inhibitory presynaptic elements.</a> J Comp Neurol. 531 (10), 1057-1079 (2023).</li><li>Yazulla, S., Studholme, K. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+miniSOG+in+the+localization+of+mitochondrial+proteins.">The use of miniSOG in the localization of mitochondrial proteins.</a> Methods Enzymol. 547, 165-179 (2014).</li><li>Shu, X., 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=A+genetically+encoded+tag+for+correlated+light+and+electron+microscopy+of+intact+cells,+tissues,+and+organisms.">A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.</a> PLoS Biol. 9 (4), e1001041 (2011).</li><li>Kar, D., 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=Volume+electron+microscopy+reveals+human+retinal+mitochondria+that+align+with+reflective+bands+in+optical+coherence+tomography+[Invited].">Volume electron microscopy reveals human retinal mitochondria that align with reflective bands in optical coherence tomography [Invited].</a> Biomed Opt Express. 14 (10), 5512-5527 (2023).</li><li>Johnson, J. 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=Spatiotemporal+regulation+of+ATP+and+Ca2++dynamics+in+vertebrate+rod+and+cone+ribbon+synapses.">Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses.</a> Mol Vis. 13, 887-919 (2007).</li></ol>

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 &#34;4F principle,&#34; which is crucial in tissue preparation. However, achi...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Electron Microscopy Science</td>
			<td>10000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit PKC</td>
			<td>Sigma-Aldrich</td>
			<td>P4334</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabbit SP</td>
			<td>Abcam</td>
			<td>ab67006</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB Substrate kit</td>
			<td>MXB Biotechnologies</td>
			<td>KIT-9701/9702/9703</td>
			<td></td>
		</tr>
		<tr>
			<td>Elbow scissors</td>
			<td>Suzhou66 vision company</td>
			<td>54010</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron microscope</td>
			<td>Phillips</td>
			<td>CM120</td>
			<td></td>
		</tr>
		<tr>
			<td>Epon resin</td>
			<td>Electron Microscopy Science</td>
			<td>14910</td>
			<td></td>
		</tr>
		<tr>
			<td>forcep</td>
			<td>Suzhou66 vision company</td>
			<td>S101A</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore filter paper</td>
			<td>Merck Millipore&#160;</td>
			<td>PR05538</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183; 12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>A component of phosphate buffer</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183; H2O</td>
			<td>Sigma</td>
			<td>71507</td>
			<td>A component of phosphate buffer</td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Science</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>VACTASTAIN ABC kit, Peroxidase (Rabbit IgG)</td>
			<td>Vector Laboratories</td>
			<td>PK-4001</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating retinal circuits and molecular localization by pre&#45;embedding immunoelectron microscopy

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.

Author Spotlight: Understanding the Ultrastructural Basis of Retinal Synaptic Connectivity and Neurotransmitter Localization in Mice 

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

In our research, we focus on retinal physiology and the disease. They need to investigate the cellular composition and the specific neurotransmitters within the retina neural circuit. The overarching goal is to explore synaptic connections and the cellular localization of neurotransmitters throughout the entire pathway.The biggest challenge of immuno electron microscope technology is difficult in achieving a balance between structure, integrity, and positive signals. Through body immunoelectron microscopy, we provide for the first time immunochemically verified ultrastructural evidence for the labs and the review and explicit distribution, novel organic cell features, and the synaptic organization or SPIR is the most All findings suggest that the crosstalk between and the raw pathway is much more extensive than previously assumed. This neurochemically identified ultrastructural results reveal that on and off signaling may interconnect through chemical synapse in the retina's OPL.

Figure 1 shows examples of control experiments without the incubation of primary antibodies against either protein kinase C alpha (PKC&#945;) or SP, in which no immunoreactivity (IR) was found.
Figure 2 depicts the PKC&#945;-IR in the mouse retina. PKC&#945; serves as a marker for all rod bipolar cells (RBC) in the retina18. At the electron microscopy (EM) level, RBC can be identified through PKC&#945;-IR, visu...

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

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-preembedding

Research

JoVE Journal

Neuroscience

131 次观看.  Wenzhou Medical University. 在我们的研究中，我们专注于视网膜生理学和疾病。他们需要研究视网膜神经回路内的细胞组成和特定神经递质。总体目标是探索突触连接和神经递质在整个通路中的细胞定位。免疫电子显微镜技术的最大挑战是难以在结构、完整性和正信号之间实现平衡。通过人体免疫电子显微镜，我们首次为实验室提供了免疫化学验证的超微结构证据，审查...