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><tbody><tr><td>1 mL syringe needle</td><td>kangdelai</td><td></td><td></td></tr><tr><td>1% OsO&lt;sub&gt;4&lt;/sub&gt;</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&amp;nbsp;</td><td>PR05538</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot; 12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71650</td><td>A component of phosphate buffer</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot; H&lt;sub&gt;2&lt;/sub&gt;O</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)&amp;nbsp;</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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

272 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 

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Medicine

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

Mouse Retina Isolation and Methanol Treatment for Studying Retinal Ganglion Cells

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

Author Spotlight: Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

Studying Bipolar and Horizontal Neuronal Cells in the Murine Split Retina

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Author Spotlight: Using the Split Retina Technique for Enhanced Access and Accelerated Experiments 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by ...

Visualizing Retinal Synaptic Structures in 3D with Volume Electron Microscopy

Preparation of Retinal Samples for Volume Electron Microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

Author Spotlight: Retinal Neuroscience Studies with Volume Electron Microscopy

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level ...

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

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

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Chapters in this video

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Preparation of Retinal Samples for Volume Electron Microscopy

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Cryosectioning and Immunostaining of Mouse Retina