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 forceps with very fine tips, scissors, a 1 mL syringe needle (needle size: G26), and filter paper.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
</ol>
2. Pre-embedding immunostaining
<ol>
	<li>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.</li>
	<li>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 &#181;m using a double-edged razor blade.</li>
	<li>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 PKC&#945;, 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 &#176;C on a shaker.</li>
	<li>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 &#176;C on a shaker. 
	NOTE: Secondary antibodies were applied alone in control experiments.</li>
	<li>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&#176;C. (Both reagent A and reagent B were diluted with 0.01 M PBS. For example: A solution: 6 &#956;l; B solution: 6 &#956;l; 0.01 M PBS: 588 &#956;l, total of 600 &#956;l).</li>
	<li>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 &#956;l; reagent 3: 50 &#956;l; distilled water: 900 &#956;l, total of 1000 &#956;l).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Post-fixation and embedding
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Put the retinal tissues in acetone solutions (50%, 70%, 80%, and 90%) for 10 min each, then in 100% twice for 10 min each.</li>
	<li>Dip the retinal tissues in a mixture containing the same volume of uranyl acetate and an epoxy resin for 1 h at 37 &#176;C drying oven, followed by a mixture containing uranyl acetate and the resin (1:4) overnight at 37 &#176;C drying oven.</li>
	<li>Transfer the retinal tissues gently using a toothpick into the new resin for 1 h at 45 &#176;C in a drying oven, and then the orientated retinal stripe is embedded in the embedding plate with the resin.</li>
	<li>Put the sample in a 45 &#176;C drying oven for 3 h and a 65 &#176;C drying oven for 48 h.</li>
	<li>Shape the embedding blocks into trapezoids and cut the block into 1 &#181;m thick sections with an ultramicrotome, stain these sections with toluidine blue, and prescreen regions of interest under a light microscope.</li>
	<li>Collect ultrathin sections (70-90 nm) on copper grids and view them under an electron microscope.</li>
</ol>

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>

The authors have no disclosures.

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.

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...

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

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

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 ...

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

Research

JoVE Journal

Neuroscience

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

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

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...