We would like to thank Bing Ye, Nathan Spix, and Hao Liu for technical support. This work was supported by the National Institute of Health Grants EY022640 (D.-Q.Z.) and Oakland University Provost Undergraduate Student Research Award (E.J.A.).

Mouse care and all experimental procedures were conducted according to the National Institutes of Health guidelines for laboratory animals and were approved by the Institutional Animal Care and Use Committees at Oakland University (protocol no. 18071).
NOTE: Names of the solutions and their compositions are listed in Table 1.
1. Tissue preparation
<ol>
	<li>Euthanize the mouse with an overdose of CO2, followed by cervical dislocation.</li>
	<li>Enucleate the eyes with curved forceps and transfer them to a small petri dish with 0.1 M PBS (Table 1). Under a dissection microscope, poke a small hole along the cornea-sclera junction with a needle. Transfer to 4% paraformaldehyde (PFA) for 1 hour.</li>
	<li>Transfer the eye back to a dish with PBS. Under a dissection microscope, use dissection scissors to cut all the way around the cornea-sclera junction. Remove the cornea and lens. Cut at the base of the optic nerve and carefully peel the sclera off with forceps to isolate the retina.</li>
	<li>Make four small cuts evenly around the retina and use a fine tip brush dipped in PBS to lay it flat (GCL side down) in a clover-like shape on a small square cut from nitrocellulose filter paper to stabilize the retina.</li>
	<li>Transfer the retina using forceps to hold the corner of the nitrocellulose paper (without touching the mounted retina) and place it in a 48-well plate with 4% PFA for 1 hour.</li>
	<li>Transfer the filter paper and retina to a well with PBS and wash (3x for 5 min each).</li>
	<li>Transfer to A4P0 (Table 1) and incubate overnight at 4 &#176;C with gentle agitation.</li>
	<li>Pipette vegetable oil into the well to completely cover the A4P0 solution. Incubate in a water bath at 40 &#176;C for 3 hours with no shaking.</li>
	<li>Wash (3x for 5 min each) in PBS, making sure all the oil has been rinsed off. If necessary, use a pipet to carefully remove remaining oil from the top of the well before the last rinse.</li>
	<li>Incubate in 10% SDS at 40 &#176;C for two days with gentle shaking. Replace SDS with fresh solution on the second day.</li>
	<li>Transfer the filter paper and retina to PBS with Triton-X-100 (PBST, Table 1) and wash (5x for 1.5 h each).</li>
	<li>Store at 4 &#176;C in PBST with 0.01% sodium azide (NaN3) or move directly to immunostaining.</li>
</ol>
2. Immunostaining and refractive index matching
<ol>
	<li>Remove the retina from the filter paper by gently peeling it off with a fine tip brush in PBST.</li>
	<li>Incubate the retina in primary antibody (Table 2) diluted in blocking solution (Table 1) for 2 days at 40 &#176;C with gentle shaking.</li>
	<li>Wash (5x, 1.5 h each) in PBST.</li>
	<li>Incubate with the appropriate secondary antibodies (Table 3) diluted in blocking solution for 2 days at 40 &#176;C with gentle shaking and protect from light through the remainder of the procedure.</li>
	<li>Wash (5x, 1.5 h each) in 0.02 M phosphate buffer (see Table 1).</li>
	<li>Incubate in sorbitol-based Refractive Index Matching Solution (sRIMS, see Table 1) at 40 &#176;C overnight with gentle shaking.</li>
</ol>
3. Mounting
<ol>
	<li>Outline a 18 mm x 18 mm x 1.5 mm glass coverslip with a fine-tip permanent marker to mark a square boundary on the back of a glass microscope slide.</li>
	<li>Flip the slide over and use a syringe to trace the boundary with a thin line of silicone grease on the front of the slide, leaving a small gap in one corner for excess mounting solution to escape.</li>
	<li>Transfer the retina to the center of the bounded area and arrange with a fine-tip brush so that it lies flat with the photoreceptor side against the glass slide.</li>
	<li>Pipette approximately 60 &#181;L of sRIMS so that it covers the flattened retina and extends to one corner of the enclosure, taking care that the retina stays flat and in place.</li>
	<li>Apply the coverslip starting from the corner with the sRIMS and slowly lower it until it touches the grease on all sides, avoiding the formation of air bubbles.</li>
	<li>Place a stack of 3 coverslips on each side of the mounted retina as a spacer. Use the long edge of another slide to press down the coverslip so that the mount is flat and even.</li>
	<li>Store slides flat at 4 &#176;C until imaging.</li>
</ol>
4. Imaging
<ol>
	<li>Image samples on either a conventional fluorescence microscope or a confocal microscope (Table of Materials). Begin by placing the slide on the microscope stage and locating the sample. 
	NOTE: If an inverted objective microscope is being used, place the slide upside down on the stage, first ensuring that the exposed areas of the slide are clear of all silicone grease and mounting solution.</li>
	<li>To obtain z-stacked images of co-labeled samples, first focus on the signal in each channel individually and set the exposure time or scanning speed, for fluorescence or confocal microscopes, respectively.</li>
	<li>Set the range for the z-stack either by manually setting the focal plane at the top and bottom of the desired range, or by setting the midpoint and then specifying a range around the midpoint.</li>
	<li>Adjust the step size or number of slices as desired.</li>
	<li>Capture the image and save the original file as well as exporting it as a TIFF file or other desired format.</li>
</ol>
5. Image analysis
<ol>
	<li>Use the image analysis software of choice (Table of Materials) to adjust the brightness and contrast in each channel until optimum clarity is achieved in both the single images and the 3-dimensional rendering of the z-stack. 
	NOTE: If the selected step size is sufficiently small, 3D deconvolution can also be performed to enhance the signal.</li>
</ol>

<ol>
	<li>Witkovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+and+retinal+function.">Dopamine and retinal function.</a> Documenta Ophthalmologica. 108 (1), 17-40 (2004).</li><li>McMahon, D. G., Iuvone, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+organization+of+the+mammalian+retina:+from+gene+regulation+to+physiology+and+diseases.">Circadian organization of the mammalian retina: from gene regulation to physiology and diseases.</a> Progress in Retinal and Eye Research. 39, 58-76 (2014).</li><li>Prigge, C. L., 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=M1+ipRGCs+Influence+Visual+Function+through+Retrograde+Signaling+in+the+Retina.">M1 ipRGCs Influence Visual Function through Retrograde Signaling in the Retina.</a> Journal of Neuroscience. 36 (27), 7184-7197 (2016).</li><li>Zhang, D. Q., Belenky, M. A., Sollars, P. J., Pickard, G. E., McMahon, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanopsin+mediates+retrograde+visual+signaling+in+the+retina.">Melanopsin mediates retrograde visual signaling in the retina.</a> PLoS One. 7 (8), 42647 (2012).</li><li>Liu, L. L., Alessio, E. J., Spix, N. J., Zhang, D. Q. <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+GluA2-containing+calcium-impermeable+AMPA+receptors+on+dopaminergic+amacrine+cells+in+the+mouse+retina.">Expression of GluA2-containing calcium-impermeable AMPA receptors on dopaminergic amacrine cells in the mouse retina.</a> Molecular Vision. 25, 780-790 (2019).</li><li>Liu, L. L., Spix, N. J., Zhang, D. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+Receptors+Contribute+to+Retrograde+Synaptic+Transmission+from+Ganglion+Cell+Photoreceptors+to+Dopaminergic+Amacrine+Cells.">NMDA Receptors Contribute to Retrograde Synaptic Transmission from Ganglion Cell Photoreceptors to Dopaminergic Amacrine Cells.</a> Frontiers in Cellular Neuroscience. 11, 279 (2017).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332 (2013).</li><li>Poguzhelskaya, E., Artamonov, D., Bolshakova, A., Vlasova, O., Bezprozvanny, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+method+to+perform+CLARITY+imaging.">Simplified method to perform CLARITY imaging.</a> Molecular Neurodegeneration. 9, 19 (2014).</li><li>Epp, J. R., 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=Optimization+of+CLARITY+for+Clearing+Whole-Brain+and+Other+Intact+Organs.">Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs.</a> eNeuro. 2 (3), (2015).</li><li>Magliaro, C., 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=Clarifying+CLARITY:+Quantitative+Optimization+of+the+Diffusion+Based+Delipidation+Protocol+for+Genetically+Labeled+Tissue.">Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue.</a> Frontiers in Neuroscience. 10, 179 (2016).</li><li>Yang, 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Zheng, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Structure and Function. 221 (4), 2375-2383 (2016).</li><li>Witkovsky, P., Arango-Gonzalez, B., Haycock, J. W., Kohler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+retinal+dopaminergic+neurons:+differential+maturation+of+somatodendritic+and+axonal+compartments.">Rat retinal dopaminergic neurons: differential maturation of somatodendritic and axonal compartments.</a> Journal of Comparative Neurology. 481 (4), 352-362 (2005).</li></ol>

The authors declare no competing financial interests.

Modification of the CLARITY protocol for whole-mount retinas. 
We have simplified the CLARITY protocol to achieve adequate polymerization without the need for a vacuum evacuation or desiccation chamber, as is used in most previous studies7,9,11. The polymerization process is inhibited by oxygen, requiring that the sample be isolated from air during the polymerization step of the protocol. However, rather th...

The vertebrate retina is perhaps the most accessible part of the central nervous system (CNS), and it serves as an excellent model for studying the development, structure, and function of the brain. Five classes of neurons in the retina are distributed in three nuclear layers separated by two plexiform layers. The outer nuclear layer (ONL) consists of classical photoreceptors (rods and cones) that convert light into electrical signals. Electrical signals are processed by neurons in the inner nuclear layer (INL), including bipolar, horizontal, and amacrine cells, and then transmitted to retinal ganglion cells (RGCs) in the ganglion cell layer (GCL). RGCs are the output neurons of the retina, with the axons projecting to the brain to contribute to image-forming and non-image-forming visual function. In addition, three types of glial cells (Muller cells, astroglia, and microglia) provide nutrients to neurons and protect neurons from harmful changes in their extracellular environment.
One specialized subpopulation of amacrine cells produces and releases dopamine, an important neuromodulator in the CNS, reconfiguring retinal neural circuits during light adaptation1,2. Dopaminergic amacrine cells (DACs) have a unique feature of morphological profiles. Their somata are located in the proximal INL with dendrites ramifying in the most distal part of the inner plexiform layer (IPL). Axon-like processes of DACs are unmyelinated, thin and long, sparsely branched, and bear varicosities (the sites of dopamine release). They form a dense plexus with dendrites in the IPL, including ring-like structures around the somata of AII amacrine cells. The axons also run through the INL toward the OPL, forming a centrifugal pathway across the retina3. We have demonstrated that DAC processes express receptors in response to glutamate release from presynaptic neurons, including bipolar cells and intrinsically photosensitive retinal ganglion cells (ipRGCs)4,5,6. However, it is unclear whether glutamate receptors express on the axons, dendrites, or both since they are cut off in vertical retinal sections and cannot be distinguished from each other5,6. Immunostaining needs to be carried out in whole-mount retinas to reveal three-dimensional branching of DACs and the presence of glutamate receptors on subcellular compartments. Although the retina is relatively transparent, the thickness of a mouse whole-mount retina is approximately 200 &#181;m, which limits the penetration of antibodies into the deep tissue as well as reduces light penetration for high-resolution imaging due to tissue light-scattering. To overcome these limitations, we adapted the immunostaining compatible tissue hydrogel delipidation method (CLARITY) developed recently for thick brain sections to whole-mount mouse retinas7.
The CLARITY method was originally developed by the Deisseroth laboratory for immunostaining and imaging of thick brain samples7. It uses a strong detergent, sodium dodecyl sulfate (SDS) and electrophoresis to remove the lipid components (that cause tissue light-scattering), leaving the proteins and nucleic acids in place. The removed lipids are replaced with a transparent scaffold made up of hydrogel monomers such as acrylamide to support the remaining protein structure. The cleared tissue can be labeled via immunohistochemistry and imaged with substantially increased light penetration depth through the tissue (up to several millimeters below the tissue surface). Since then, the CLARITY method has been optimized and simplified by several research groups8,9,10. A modified CLARITY protocol uses a passive clearing technique to avoid the possible tissue damage produced by electrophoresis for clearing the whole-brain and other intact organs11. However, this method has not yet been applied to whole-mount retinas. Here, we adapted the passive CLARITY technique for whole-mount retinas to make them more transparent for immunohistochemistry and imaging. We found that a majority of the retinal proteins tested were preserved during this process for immunohistochemistry. Using the refractive index matching solution, we were able to image retinal neurons across the approximately 200 &#181;m thickness from the ONL to the GCL in whole-mount retinas.

<table><tbody><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Fixative</td></tr><tr><td>Acrylamide</td><td>Fisher Biotech</td><td>BP170</td><td>Hydrogel monomer</td></tr><tr><td>Axio Imager.Z2</td><td>Zeiss</td><td></td><td>Fluorscence microscope</td></tr><tr><td>BSA</td><td>Fisher Scientific</td><td>BP1600</td><td>Blocking agent</td></tr><tr><td>Eclipse Ti</td><td>Nikon Instruments</td><td></td><td>Scanning confocal microscope</td></tr><tr><td>KCl</td><td>VWR</td><td>BDH0258</td><td>Buffer component</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td>Buffer component</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S9763</td><td>Buffer component</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td>Buffer component</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S0751</td><td>Buffer component</td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S2002</td><td>Bacteriostatic preservative</td></tr><tr><td>NDS</td><td>Aurion</td><td>900.122</td><td>Blocking agent</td></tr><tr><td>NIS Elements AR</td><td>Nikon</td><td></td><td>Image analysis software</td></tr><tr><td>SDS</td><td>BioRad</td><td>1610301</td><td>Delipidation agent</td></tr><tr><td>Sorbitol</td><td>Sigma Aldrich</td><td>51876</td><td>Buffer component</td></tr><tr><td>Triton-X-100</td><td>Sigma</td><td>T8787</td><td>Surfactant</td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337</td><td>Surfactant</td></tr><tr><td>VA-044</td><td>Wako Chemicals</td><td>011-19365</td><td>Thermal initiator</td></tr></tbody></table>

immunostaining of whole-mount retinas with the clarity tissue clearing method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Modified CLARITY-processed retinas are optically transparent tissue. 
To formulate a tissue clearing method that is compatible with immunohistochemical applications in the retina while providing adequate delipidation and retaining the structural integrity of the cellular proteins, we adapted the CLARITY tissue clearing method to whole-mount mouse retinas. We were able to simplify the protocol and modify it for whole-mount retinas (see Protocol). After completing tissue hybridization, clearing, and r...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method at JoVE.com

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

immunostaining-whole-mount-retinas-with-clarity-tissue-clearing

Research

JoVE Journal

Neuroscience

7.4K Views.  Oakland University. Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

Video: Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; 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.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

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

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Medicine

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

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

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

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 pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

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 ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...