Name: Preparation of Retinal Samples for Volume Electron Microscopy (Video) | JoVE Journal
Uploaded: 2024-05-24T13:50:00.000+00:00
Duration: 3 min 48 s
Description: Learn retinal sample preparation for volume electron microscopy using FIB-SEM to examine nanoscale 3D structures of photoreceptor terminals in mouse retina. This optimized protocol enables detailed reconstruction of synaptic architecture.

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5 minutes

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

Animal care and use protocols were approved by the Ethics Committee of Wenzhou Medical University and followed the guidelines established by the Association for Research in Vision and Ophthalmology (ARVO). All mice were maintained in a 12-h light and 12-h dark cycle and supplied with a standard chow diet.
1. Retina dissection and fixation
<ol>
	<li>Anesthetize the mice (C57BL/6J, Male, 8-week-old, 20-25 g) by injecting 2,2,2-tribromoethanol (0.25 mg/g of body weight) intraperitoneally. Confirm the depth of anesthetization by no retraction of the back paw after pinching the toe or no blinking reflex. Then, dislocate the cervical vertebra while still under anesthesia to euthanize the animal. 
	NOTE: Tribromoethanol was chosen for its well-documented efficacy in inducing rapid and reliable anesthesia in small animal models, particularly for short-term procedures. While pharmaceutical-grade anesthetics are preferred, tribromoethanol offers specific advantages in certain protocols, making it a suitable alternative when properly prepared and administered. All precautions were taken to ensure its safe and effective use. This study adheres to ethical considerations by ensuring proper preparation, administration, and monitoring to mitigate potential risks associated with its use.</li>
	<li>Remove the eyes with curved scissors and immerse the eyes in a mixed solution containing 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4).</li>
	<li>Remove the anterior segment and vitreous from the eyes with forceps under the dissecting microscope.</li>
	<li>Peel the sclera with the two forceps until the retina is completely isolated from the eyecup.</li>
	<li>Cut the retina rapidly into 100 - 200 &#181;m-thick strips with a razor and handle the retina strips with a Pasteur pipette into a new microcentrifuge (EP) tube containing 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4) for 2 h at room temperature (RT) on the rotator. After completion, place the tubes to fix at 4 &#176;C for 24 h.</li>
</ol>
2. Sample embedding process
<ol>
	<li>After washing in 0.1 M sodium cacodylate buffer (pH 7.4) 5 times&#160;for 15 min each, incubate the retina strips with a solution containing 1.5% potassium ferrocyanide and 1% OsO4(in PB, pH7.4) in 0.1 M sodium cacodylate with 4 mM CaCl2 for 1.5 h in the dark on ice. 
	NOTE: Dark incubation and lack of phosphate can prevent precipitation and artifacts in the sample.</li>
	<li>Wash the retina strips 3 times&#160;in ddH2O for 10 min each, and then treat with 1% thiocarbohydrazide in ddH2O for 30 min in the dark at RT.</li>
	<li>After rinsing in ddH2O 3 times&#160;for 15 min each, incubate the retina strips in 2% OsO4 (in PB, PH 7.4) for 45 min in the dark at RT.</li>
	<li>After washing in ddH2O 3 times&#160;for 15 min each, immerse the retina strips in 1% aqueous uranyl acetate in the dark at 4 &#176;C&#160;overnight.</li>
	<li>Next day, after washing in ddH2O 3 times&#160;for 15 min each, treat the retina strips with 0.66% lead nitrate in L-aspartic acid (0.03 M, pH 5.5) for 40 min at 65 &#176;C.</li>
	<li>After washing in ddH2O 3 times&#160;for 15 min each, dehydrate the retina strips with ascending ethanol concentration of 30%, 50%, 70%, 90%, and 100% for 20 min in each solution.</li>
	<li>Treat retina strips with a mixed solution containing equal ethanol and acetone for 20 min, followed by an acetone wash 2 times&#160;for 20 min each.</li>
	<li>Infiltrate the retina strips in acetone/resin mixed in a ratio of 7:3 in the dark at RT&#160;overnight and then acetone/resin at 3:7 in the dark for 24 h at RT. 
	NOTE: The precise composition of the resin is as follows: Epon812 10.06 mL, DDSA 4 mL, MNA 5.94 mL, and DMP-30 0.2 mL.</li>
	<li>Change the tubes the next day and infiltrate the retina strips with pure resin for 24 h at 45 &#176;C. Change the resin again the next day, and embed the retina strips for 48 h at 60 &#176;C. 
	NOTE: The purpose of increasing the temperature is to improve penetration.</li>
</ol>
3. Selection of the area of interest
<ol>
	<li>Cut the resin blocks into 1 &#181;m thick sections, stain sections with 1% toluidine-blue, and observe the general structure of the retina under light microscopy to select the rough regions of interest.</li>
	<li>Coat the resin blocks with platinum using a sputter coater, followed by milling with 70 nm thick section using a focused-ion-beam scanning electron microscopy under a current of 0.23 nA and acceleration voltage of 30 kV.</li>
	<li>Pre-screen the section using FIB-SEM with 2000x magnification to identify the precise regions of interest.</li>
	<li>Collect the data using FIB-SEM with a beam current of 0.69 nA and acceleration voltage of 2 kV. The other parameters are as follows: dwell time = 2 &#181;s, voxel size = 1.8 nm x 1.8 nm x 15 nm.</li>
</ol>

Visualizing Retinal Synaptic Structures in 3D with Volume Electron Microscopy

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<li>Schmitz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+making+of+synaptic+ribbons%3A+how+they+are+built+and+what+they+do%5BTitle%5D)+AND+%22Neuroscientist%22%5BJournal%5D)">The making of synaptic ribbons: how they are built and what they do.</a> Neuroscientist. 15 (6), 611-624 (2009).</li>
<li>Kantardzhieva, A., Peppi, M., Lane, W. S., Sewell, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+composition+of+immunoprecipitated+synaptic+ribbons%5BTitle%5D)+AND+%22J+Prot+Res%22%5BJournal%5D)">Protein composition of immunoprecipitated synaptic ribbons.</a> J Prot Res. 11 (2), 1163-1174 (2011).</li>
<li>Zampighi, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conical+tomography+of+a+ribbon+synapse%3A+structural+evidence+for+vesicle+fusion%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Conical tomography of a ribbon synapse: structural evidence for vesicle fusion.</a> PLoS One. 6 (3), e16944(2011).</li>
<li>Barnes, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+diversity+of+the+rod+spherule%3A+A+study+of+serially+reconstructed+electron+micrographs%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">Morphological diversity of the rod spherule: A study of serially reconstructed electron micrographs.</a> Plos One. 11 (3), e0150024(2016).</li>
<li>Xu, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+FIB-SEM+systems+for+large-volume+3D+imaging%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Enhanced FIB-SEM systems for large-volume 3D imaging.</a> Elife. 6, e25916(2017).</li>
<li>Crewe, A. V., Lin, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+backscattered+electrons+for+imaging+purposes+in+a+scanning+electron+microscope%5BTitle%5D)+AND+%22Ultramicroscopy%22%5BJournal%5D)">The use of backscattered electrons for imaging purposes in a scanning electron microscope.</a> Ultramicroscopy. 1 (3), 231-238 (1976).</li>
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<li>Wergin, W. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+thin+and+thick+sections+of+biological+tissue+with+the+secondary+electron+detector+in+a+field-emission+scanning+electron+microscope%5BTitle%5D)+AND+%22Scanning%22%5BJournal%5D)">Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope.</a> Scanning. 19 (6), 386-395 (1997).</li>
<li>Willingham, M. C., Rutherford, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+osmium-thiocarbohydrazide-osmium+%28OTO%29+and+ferrocyanide-reduced+osmium+methods+to+enhance+membrane+contrast+and+preservation+in+cultured+cells%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.</a> J Histochem Cytochem. 32 (4), 455-460 (1984).</li>
<li>Buravkov, S. V., Chernikov, V. P., Buravkova, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+method+of+specimen+preparation+for+scanning+electron+microscopy%5BTitle%5D)+AND+%22Bull+Exp+Biol+Med%22%5BJournal%5D)">Simple method of specimen preparation for scanning electron microscopy.</a> Bull Exp Biol Med. 151 (3), 378-382 (2011).</li>
<li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+diversity+in+the+retina%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Neuronal diversity in the retina.</a> Curr Opin Neurobiol. 11 (4), 431-436 (2001).</li>
<li>Svara, F. N., Kornfeld, J., Denk, W., Bollmann, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volume+EM+reconstruction+of+spinal+cord+reveals+wiring+specificity+in+speed-related+motor+circuits%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Volume EM reconstruction of spinal cord reveals wiring specificity in speed-related motor circuits.</a> Cell Rep. 23 (10), 2942-2954 (2018).</li>
<li>Kubota, Y., Sohn, J., Kawaguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large+volume+electron+microscopy+and+neural+microcircuit+analysis%5BTitle%5D)+AND+%22Front+Neural+Circuits%22%5BJournal%5D)">Large volume electron microscopy and neural microcircuit analysis.</a> Front Neural Circuits. 12, 98(2018).</li>
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The authors have no disclosures.

We implemented the OTO&#39;s Volume EM sample preparation protocol to analyze the photoreceptors&#39; terminal structure in retinal tissue. The focus was on detailing the entire procedure, starting from the detachment and fixation of the retina to showcasing the results of 3D reconstruction of photoreceptor axon terminals.
The distinctive feature of retinal tissue, unlike brain tissue, lies in its lack of regional differences. Comprising three layers of neuronal cell bodies and two layers of synaptic connections, the retina exhibits a regular and hierarchical structure despite the complexity of its neuronal and synaptic components20. In dissecting the retina, it becomes imperative to ensure that the thin strips are minimized to guarantee uniform contrast in subsequent sample preparation stages. The photoreceptor axon terminals, critical structures of interest, are situated in the outer plexiform layer (OPL) of the retina. The hierarchical and organized nature of the retina allows for efficient localization of the target structures, facilitating precise sampling during EM and ensuring the accuracy of subsequent 3D reconstruction results.
Volume EM facilitates three-dimensional visualization of local neural circuits and synaptic connections within the retina at the nanoscale, utilizing serial sections21,22. This technique enables the automatic generation of high-resolution 3D images, and FIB-SEM holds the potential to unveil intricate details of both physiological and pathological activities. Therefore, maintaining stable image contrast throughout the entire block is crucial during the sample preparation process. To achieve uniform conductivity, we employ the pre-embedding OTO method, a classical approach widely acknowledged for its effectiveness in enhancing contrast in scanning electron microscopy (SEM)18,23,24. This method involves a double osmium coating combined with thiocarbohydrazide, ensuring optimal bulk conductivity for large samples. Additionally, the double osmium treatment contributes to the superior preservation of lipid membrane structures, adding a layer of precision to our investigations. The retinal samples are hierarchical, the tissue blocks are small and easy to penetrate, and the OTO method can maintain better cell membrane contour and uniform image contrast.
Several critical factors contribute to the success of this protocol. Firstly, swift retinal harvesting and precise fixation times are paramount for maintaining optimal subcellular structure. Secondly, ensuring appropriate concentrations of osmium and thiocarbohydrazide is essential to preserve contrast in the membrane structure of the series images. Osmium, in particular, plays a crucial role by binding with lipid molecules, enhancing the BSE signal.
Our retinal sample preparation protocol is user-friendly, requires no additional equipment, and minimizes processing time. This characteristic highlights its potential applicability to other Volume EM techniques, suggesting versatility and ease of adoption in diverse experimental setups. The OTO&#39;s method notably enhances the contrast of FIB-SEM images. However, the details about the cell membrane, especially the bilayer structure, may still appear somewhat blurred. Our current sample preparation scheme involves some sacrifice of small details in cell structure to a certain extent. We aim to develop optimized methods in the future to address and improve this limitation.

The retina is densely packed with intertwining neuronal axons and dendrites that form synapses between them1. Microscopy is an indispensable tool for studying retinal anatomy as it has fine, intricate, and small structures. Although electron microscopy (EM) provides unparalleled power to investigate the ultrastructure of subcellular organelles and the accurate localization of specific proteins at the nanometer level2, it produces images limited to the two-dimensional (2D) plane, leading to potential loss of key information.
The development of emerging high-resolution volume electron microscopy (Volume EM) techniques supports the provision of more comprehensive and larger-scale three-dimensional (3D) structural information. Some 3D EM methods have been recently reviewed by others3,4,5. 3D EM allows for the reconstruction of neuronal shape and connectivity details, enabling precise quantitative analysis of structures of interest. This demonstrates that the data obtained by volume EM are more systematic, complete, and accurate.
Retinal photoreceptors, constituting the initial neurons in visual signaling6,7, establish synapses with dendrites of second-order bipolar and horizontal cells in the photoreceptor&#39;s terminal to facilitate excitatory signals8,9. These terminals, referred to as cone pedicles and rod spherules, encompass three crucial components: mitochondria, synaptic ribbons, and synaptic vesicles. While previous studies have predominantly concentrated on the general structure of ribbon synapses, there has been a notable absence of investigation into the fine structure of major components, including mitochondria, ribbon, vesicle pools, and their spatial organization in terminals10,11,12,13. A precise and systematic analysis of each component, along with an understanding of their inter-association within photoreceptor terminals, is vital for unraveling spatial organization and comprehensively grasping visual processing functions. In photoreceptors, mitochondria are mainly present in the inner segment, cell body, and terminal. We focused here on the mitochondria in the terminal photoreceptors. Focused ion beam scanning electron microscopy (FIB-SEM), a type of volume EM boasting high resolution (x, y, and z resolution &#60; 5 nm) and a relatively large volume flux4,14, stands as a potent tool for accurately visualizing the 3D structure of photoreceptor terminals.
Both FIB-SEM and Serial Block Face Scanning Electron Microscope (SBF-SEM) are Volume EM based on SEM for obtaining the image of tissues by scanning the surface of the sample. The ultrastructural features of a specimen&#39;s surface are revealed through the contrast created by the intensity of secondary or backscattered electrons (BSE) when the electron beam scans the sample15. Essentially, detecting BSE or secondary electrons from the cross-section surface of a resin-embedded tissue sample in SEM allows for obtaining images of the embedded sample16,17. When BSE or secondary electrons are less generated, information on the sample surface can only be obtained. Achieving consistent contrast and high-quality serial images necessitates sufficient deposition of heavy metals in the sample. Therefore, specific sample preparation protocols are crucial for subsequent segmentation, 3D reconstruction, and analysis when utilizing SEM for serial imaging. The osmium-thiocarbohydrazide-osmium (OTO) method is a typical sample preparation scheme for 3D electron microscopy of biological samples, preserving the structure of lipid-containing membranes and maintaining good contrast18,19.
Here, we developed the OTO method for the preparation of retinal samples for the use of Volume EM. This process particularly focuses on dissecting the retina, determining the optimal fixation time for retinal tissue, and detailing the specific procedures and precautions in 3D sample preparation. Additionally, segmentation and 3D reconstruction of the target structure are integral steps in this extended application. The retina, being a small and challenging structure to obtain materials from, requires swift and precise operations for EM, with fixed times and fresh reagents prepared for immediate use.

<table><tbody><tr><td>2,2,2-Tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402</td><td></td></tr><tr><td>Acetone</td><td>Electron Microscopy Science</td><td>10000</td><td></td></tr><tr><td>Amira 6.8</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C-2661</td><td></td></tr><tr><td>Embedding mold</td><td>Beijing Zhongjingkeyi Technology</td><td>GP10590</td><td></td></tr><tr><td>Epon resin</td><td>Electron Microscopy Science</td><td>14900</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>64-17-5</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Electron Microscopy Science</td><td>16020</td><td></td></tr><tr><td>Helios NanoLab 600i dual-beam SEM</td><td>FEI</td><td></td><td></td></tr><tr><td>L-aspartic acid</td><td>Sigma</td><td>56-84-8</td><td></td></tr><tr><td>Lead nitrate</td><td>Sigma</td><td>10099-74-8</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.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;.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>OsO&lt;sub&gt;4&lt;/sub&gt;</td><td>TED PELLA</td><td>4008-160501</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Science</td><td>157-8</td><td></td></tr><tr><td>Potassium ferrocyanide</td><td>Sigma</td><td>14459-95-1</td><td></td></tr><tr><td>Sodium cacodylate</td><td>Sigma</td><td>6131-99-3</td><td></td></tr><tr><td>Sputter coater</td><td>Leica</td><td>ACE200</td><td></td></tr><tr><td>Thiocarbohydrazide</td><td>Sigma</td><td>2231-57-4</td><td></td></tr><tr><td>Uranyl acetate</td><td>TED PELLA</td><td>CA96049</td><td></td></tr></tbody></table>

preparation of retinal samples for 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.

Figure 1A shows the image of retinal photoreceptor terminals prepared using the traditional chemical double fixation method, and Figure 1B shows the image of retinal photoreceptor terminals prepared using the OTO method. Both were sampled by FIB-SEM. It can be clearly seen that the cell membrane structure can be retained as much as possible by using the OTO method, and even the outline of vesicles can be clearly seen. In addition, the contrast of images obtained by using the OTO method is also clearer.
Figure 2 illustrates the experimental workflow employed for reconstructing the ultrastructural characteristics of photoreceptor terminals. The reconstruction focused on four types of photoreceptor terminals, including M-cone pedicles, S-cone pedicles, rod1 spherules, and rod2 spherules. The main emphasis was on essential components, such as mitochondria, synaptic ribbons, and synaptic vesicle pools within the photoreceptor terminals.
Figure 3 displays the reconstructed mitochondria and synaptic ribbons in the rod photoreceptor terminals. Notably, the synaptic ribbons of the rod2 spherules exhibit a distinctive horseshoe shape, typically featuring a single ribbon per spherule. Moreover, Figure 2 highlights the presence of two mitochondria in rod spherules, with a consistent position within the central space of the terminals, located above the synaptic ribbons. All the structures were traced manually, page by page.
<img alt="Electron microscopy images, A-B, cell ultrastructure comparison, cellular organelles, high magnification." class="xfigimg" src="/files/ftp_upload/66589/66589fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Examples of images sampled using FIB-SEM for traditional chemical double fixation method and OTO method. (A) Traditional chemical double fixation method. (B) OTO method. Scale bar: 1000 nm. <a href="https://www.jove.com/files/ftp_upload/66589/66589fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electron microscopy image processing: alignment, segmentation, 3D render, cellular structure analysis." class="xfigimg" src="/files/ftp_upload/66589/66589fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: 3D reconstruction workflow for photoreceptor terminals using FIB-SEM. Following alignment using 3D data visualization software, the serial images were manually segmented and rendered for further analysis. (A) Original serial images. (B) Aligned images. (C) Segmented images. (D) Reconstructed images. <a href="https://www.jove.com/files/ftp_upload/66589/66589fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cellular structure illustration; nanoscale imaging; highlights subcellular components; microscopy." class="xfigimg" src="/files/ftp_upload/66589/66589fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: 3D reconstruction. The representative 3D reconstruction images depict photoreceptor terminals in white, mitochondria in orange, and synaptic ribbons in blue. Scale bar: 500 nm. <a href="https://www.jove.com/files/ftp_upload/66589/66589fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

preparation-of-retinal-samples-for-volume-electron-microscopy

Research

JoVE Journal

Neuroscience

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.

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

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.

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

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

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.

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor β after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Learn retinal sample preparation for volume electron microscopy using FIB-SEM to examine nanoscale 3D structures of photoreceptor terminals in mouse retina. This optimized protocol enables detailed reconstruction of synaptic architecture.

Preparation of Retinal Samples for Volume Electron Microscopy

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