This work was supported by NIH grant R01-EY020535 (B.T.S), International Retinal Research Foundation and Karl Kirchgessner Foundation (B.T.S).

In all experimental procedures, animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with protocols approved by Weill Cornell Medical College. Animals were euthanized by carbon dioxide and subsequent cervical dislocation.
1. Dissection of Wholemount Retina
<ol>
	<li>After the mouse is euthanized, enucleate its eyes and place them into oxygenated HEPES-buffered Ringer&#39;s solution (Table 1).</li>
	<li>Remove the cornea by cutting in a circular path along the ora serrata with small scissors, holding the eye at the limbus with forceps. Pull out the lens with forceps.</li>
	<li>Remove as much vitreous as possible, using blunted-edge forceps. This step is important for bolus injections and electrophysiological recordings; it is not necessary for when performing immunohistochemistry alone.</li>
	<li>Detach the retina from the eyecup by positioning forceps between the retina and the eyecup, moving the forceps slowly around the circumference. Stabilize the eye cup by holding the sclera firmly with a second set of forceps. Cut the optic nerve at the location between the retina and the eyecup to ease the separation of the retina from the eyecup.</li>
	<li>The retina can be dissected into pieces (e.g. quarters) or used as a whole. To cut into pieces, use scissors to make incisions from the retinal periphery all the way down to the optic nerve. Alternatively, to use the entire retina, make incisions from the periphery halfway to the optic nerve. The incisions will help to make a flat preparation.</li>
</ol>
2. Mounting onto Hydrophilized PTFE Membrane Insert
<ol>
	<li>Prepare the membrane as described below. When physiological recordings or dye injection experiments are performed, shorten the holder by using either a lathe or a handsaw so that the remaining ring has a height of 1 mm. 
	Note: The culture plate inserts may be either unmodified or modified (Figure 1A). The choice of the configuration is dictated by the purpose of the experiment. For immunostaining, an unmodified insert is suitable.</li>
	<li>Polish down rough surfaces with fine (300 grit or higher) sandpaper.</li>
	<li>(Optional) Remove the feet underneath the ring with scissors.</li>
	<li>To remove any debris, rinse or sonicate the membrane in distilled water for 2-3 min.</li>
	<li>Mount the tissue onto the membrane (Figure 1A). Place the tissue with some solution on top of the membrane with a plastic transfer pipette. Cut off the narrow tip of the pipette if necessary, to avoid having the tissue sample adhere to the pipette during transfer.</li>
	<li>Attach the retina with ganglion cells (GCs) up: on the membrane, gradually remove solution while spreading the retina and unfolding the folds with forceps.</li>
	<li>On the opposite side of the membrane, position an empty syringe (1 ml) directly underneath the tissue. Apply suction by pulling back on the plunger to a volume of 0.2-0.3 ml. Increase the surface area of suction by cutting the holding tip of the syringe prior to use. The tissue does not require further treatment for adhesion, as it is physically enmeshed within the membrane.</li>
</ol>
3. Bolus Injection of Isolectin Alexa 488
<ol>
	<li>Prepare patch pipettes and staining solution. Pull patch pipettes from borosilicate capillary glass with a Flaming/Brown puller, with a resistance of ~1 M&#8486;. Gently break the tip of the pipette.</li>
	<li>Make staining solution by adding 0.3 &#956;l of isolectin Alexa 488 stock solution to 10 &#956;l of the HEPES-buffered Ringer&#39;s solution described in step 1.2.</li>
	<li>Transfer sample to the patch-clamp setup for injection (Figure 1C). Place the membrane with attached tissue into the recording chamber perfused with warmed (37 &#176;C) and oxygenated HEPES-buffered Ringer&#39;s solution.</li>
	<li>Place the pipette containing dye against the inner limiting membrane (ILM, Figure 2A). Using micromanipulator, further advance the pipette against the ILM until it penetrates it. Inject the staining solution with a pressure 10-20 psi for 1 sec using a picospritzer. Single or multiple locations may be targeted for injection (Figure 2B). If a picospritzer is not available, a 10 ml syringe can be used to deliver a positive air pressure for injection.</li>
	<li>Incubate injected tissue for 10 min. During incubation, the injected tissue can be viewed under epifluorescence illumination.</li>
	<li>After labeling, the preparation may be taken to a microscope for imaging. Transfer the insert to a glass-bottom culture dish, placing retina side down (Figure 1C).</li>
	<li>Add a drop of HEPES solution to keep the specimen moist.</li>
	<li>Place 2-3 spots of vacuum grease around the perimeter of the plastic ring to firmly secure the insert to the bottom of the culture dish.</li>
	<li>Transfer the dish to the microscope stage.</li>
</ol>
4. Paraformaldehyde and Carbodiimide Fixation
<ol>
	<li>Fix the sample by transferring the insert into fixative for 15 min. Longer or shorter fixation times may be used, dependent on application. Both fixatives can be effectively used to fix GFP, isolectin, or fluorescent Alexa hydrazides (e.g. used for filling the cells in patch-clamp recordings). 
	Note: For fixation, either 4% carbodiimide or 4% paraformaldehyde solutions may be used. To maintain adherence, fixation should be done after the tissue has already been attached to the membrane. The tissue can be fixed immediately after mounting, or after physiological recording and/or bolus injection.</li>
	<li>Rinse the tissue in phosphate buffer (PB) for 30 min. After this, the tissue may be processed either as a wholemount (step 5) or cut into vertical sections and then processed (steps 6 and 7).</li>
</ol>
5. Immunostaining of Retinal Wholemounts
<ol>
	<li>In a 24-well dish (Figure 1D) block retinal pieces on the membrane for 1 hr in a staining solution.</li>
	<li>Dilute mouse anti-PSD-95 primary antibodies 1:300 in the staining solution and apply for 48 hr at room temperature in dark. Add ~100-150 &#956;l antibody solution in each well with the tissue. To prevent drying out, fill unused wells with distilled water.</li>
	<li>Rinse for 20 min in PB, 3x.</li>
	<li>Incubate for 24 hr in donkey anti-mouse Alexa 568 secondary antibody dissolved 1:500 in the staining solution.</li>
	<li>Rinse for 20 min in PB, 3x.</li>
	<li>Place the insert with the stained tissue into a standard 3 cm Petri dish filled with PB solution. With small scissors, cut out the portion of the membrane containing the retina from the ring.</li>
	<li>Transfer the retina on the membrane onto a slide retina up. Remove PB, add mounting medium. Place small pieces of a broken cover slip glass in between the slide and the coverslip glasses to avoid extensive squeezing and damage to the retina. Place coverslip.</li>
	<li>Seal the coverslip in place with nail polish. Keep slides in the dark, in a refrigerator.</li>
</ol>
6. Vertical Sectioning of Membrane-mounted Tissue
<ol>
	<li>After fixation, submerse the tissue on the membrane into the sucrose gradient solutions of 10%, 20%, and 30% sucrose for 30 min, 1 hr, and overnight, respectively. The overnight step is performed at 4 &#176;C in refrigerator, all other steps at room temperature.</li>
	<li>Using scissors, remove the membrane with the tissue from the plastic insert ring.</li>
	<li>Place the cut off membrane onto Parafilm with retina up and remove as much solution as possible with Kimwipes.</li>
	<li>Put the Parafilm with the tissue into the cryostat chamber and add a drop of OCT medium. Use the quick freezer device to accelerate the freezing.</li>
	<li>Detach the frozen sample and flip it over.</li>
	<li>Cut excess frozen OCT. Leave approximately 2 mm around the sample on three sides, and leave 5-6 mm from the side which will be attached to the holder.</li>
	<li>Cover exposed retinal side (membrane side) with a drop of OCT. Leave the attaching area free.</li>
	<li>Freeze the membrane with retinal tissue. Now the sample is ready for placing onto a cutting holder.</li>
	<li>Place a drop of OCT onto a cold holder ring. Immediately insert the frozen sample into the drop.</li>
	<li>Cut sections 10-20 &#956;m thick, setting cryostat temperature at -19 &#176;C. Use warm (room temperature) polysine adhesion slides. Leave the slides with the attached sections onto a heating platform for ~2-3 min (41 &#176;C) in dark to ensure firm adhesion of sections.</li>
	<li>Put the slides into a storage box and keep at -20 &#176;C until use.</li>
</ol>
7. Immunostaining of Retinal Sections
<ol>
	<li>Take the slides with the sections out of the freezer. Let them stay at room temperature for 1 min to remove the condensate.</li>
	<li>Encircle the sections with a liquid blocker to create a barrier for further incubation.</li>
	<li>Place the slides into the incubation box. Cover the bottom of the box with water to prevent evaporation of the antibody solution.</li>
	<li>Rinse with PB for 5 min.</li>
	<li>Prepare the same staining solutions and follow the steps as in step 5. The incubation with the primary antibody is overnight and with the secondary 1 hr. Do not use broken glass between the coverslip and the slide.</li>
</ol>

<ol>
	<li>Csaba, G., Kovacs, P., Pallinger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDAC+fixation+increases+the+demonstrability+of+biogenic+amines+in+the+unicellular+Tetrahymena:+a+flow+cytometric+and+confocal+microscopic+comparative+analysis.">EDAC fixation increases the demonstrability of biogenic amines in the unicellular Tetrahymena: a flow cytometric and confocal microscopic comparative analysis.</a> Cell Biol. Int. 30, 345-348 (2006).</li><li>Fletcher, E. L., Hack, I., Brandstatter, J. H., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+localization+of+NMDA+receptor+subunits+in+the+rat+retina.">Synaptic localization of NMDA receptor subunits in the rat retina.</a> J. Comp. Neurol. 420, 98-112 (2000).</li><li>Gastinger, M. J., O'Brien, J. J., Larsen, N. B., Marshak, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+immunoreactive+axons+in+the+macaque+retina.">Histamine immunoreactive axons in the macaque retina.</a> Invest. Ophthalmol. Vis. Sci. 40, 487-495 (1999).</li><li>Haverkamp, S., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+the+mouse+retina.">Immunocytochemical analysis of the mouse retina.</a> J. Comp. Neurol. 424, 1-23 (2000).</li><li>Koulen, P., Fletcher, E. L., Craven, S. E., Bredt, D. S., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+localization+of+the+postsynaptic+density+protein+PSD-95+in+the+mammalian+retina.">Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina.</a> J. Neurosci. 18, 10136-10149 (1998).</li><li>Pena, J. T., 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=miRNA+in+situ+hybridization+in+formaldehyde+and+EDC-fixed+tissues.">miRNA in situ hybridization in formaldehyde and EDC-fixed tissues.</a> Nat. Methods. 6, 139-141 (2009).</li><li>Tymianski, 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=A+novel+use+for+a+carbodiimide+compound+for+the+fixation+of+fluorescent+and+non-fluorescent+calcium+indicators+in+situ+following+physiological+experiments.">A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.</a> Cell Calcium. 21, 175-183 (1997).</li><li>Sagdullaev, B. T., Eggers, E. D., Purgert, R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+interactions+between+excitatory+and+inhibitory+retinal+synapses+control+visual+output.">Nonlinear interactions between excitatory and inhibitory retinal synapses control visual output.</a> J. Neurosci. 31, 15102-15112 (2011).</li><li>Toychiev, A. H., Sagdullaev, B., Yee, C. W., Ivanova, E., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time+and+cost+efficient+approach+to+functional+and+structural+assessment+of+living+neuronal+tissue.">A time and cost efficient approach to functional and structural assessment of living neuronal tissue.</a> J. Neurosci. Methods. 214, 105-112 (2013).</li><li>Yee, C. W., Toychiev, A. H., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+deficiency+exacerbates+impairment+in+a+mouse+model+of+retinal+degeneration.">Network deficiency exacerbates impairment in a mouse model of retinal degeneration.</a> Front Syst. Neurosci. 6, 8 (2012).</li><li>Ivanova, E., Muller, U., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+glycinergic+input+to+bipolar+cells+of+the+mouse+retina.">Characterization of the glycinergic input to bipolar cells of the mouse retina.</a> Eur. J. Neurosci. 23, 350-364 (2006).</li><li>Dmitrieva, N. A., Strang, C. E., Keyser, K. T. <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+alpha+7+nicotinic+acetylcholine+receptors+by+bipolar,+amacrine,+and+ganglion+cells+of+the+rabbit+retina.">Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina.</a> J. Histochem. Cytochem. 55, 461-476 (2007).</li></ol>

The authors have no competing financial interests.

The hydrophilized PTFE membrane&#39;s high biocompatibility and transparency in solutions is advantageous when working with living tissue. This preparation has been successfully used in earlier work for patch-clamp recordings of light responses8-10. Here, we show how this approach can be adopted for structural analysis of the wholemount retina.
The bolus loading technique can be effectively used for labeling living tissue. This technique is aided by the structural support of the mem...

Performing immunohistochemistry in delicate intact tissues runs the risk of damage during handling and transfer. This can occur either in brain slices or other thin tissue, such as degenerated retina. Additionally, there are certain methods of tissue fixation that can be advantageous for immunostaining of neuronal structures, but result in compromised structural stability, precluding their use. A particular example of this is carbodiimide-based fixation, which is superior for staining receptors and hormones1-7 but is commonly avoided due to the instability of the fixed tissue.
Here, we describe a procedure which utilizes a hydrophilized PTFE membrane to structurally support delicate tissue, either fixed or unfixed, for a variety of staining techniques. The supporting membrane allows for the manipulation of delicate tissue before and after fixation, allowing for several steps of processing while minimizing the risk of tissue damage. Overall, this simple method to preserve tissue integrity provides the opportunity to use techniques that might otherwise be avoided. As such, this approach could also be successfully used for preparation of wide variety of tissues such as brain slices that become fragile following slicing procedures.

<table><tbody><tr><td>Millicell Cell Culture Insert, 12 mm,&lt;br/&gt; hydrophilic PTFE (Biopore), 0.4 &amp;mu;m</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>Insulin syringe, 1 ml</td><td>Beckton Dickinson</td><td>309659</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>15003-08</td><td>dissection</td></tr><tr><td>Forceps, Dumont #55, inox</td><td>Fine Science Tools</td><td>11255-20</td><td>dissection</td></tr><tr><td>Cryostat</td><td>Leica</td><td>various</td><td></td></tr><tr><td>Confocal Microscope System</td><td>Nikon</td><td>various</td><td></td></tr><tr><td>Capillary glass</td><td>World Precision Instruments</td><td>1B150F-4</td><td></td></tr><tr><td>P-97 Flaming/Brown Micropipette Puller</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Picospritzer III</td><td>Parker Hannifin</td><td></td><td></td></tr><tr><td>Glass Bottom Culture Dishes</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Petri dish</td><td>Falcon</td><td>1008</td><td></td></tr><tr><td>Disposable Graduated Transfer Pipettes</td><td>VWR</td><td>16001-180</td><td></td></tr><tr><td>Multiwell plates, 24-well</td><td>Beckton Dickinson</td><td>351147</td><td></td></tr><tr><td>Cover glass, #1</td><td>Electron Microscopy Sciences</td><td>72200-30</td><td></td></tr><tr><td>Polysine adhesion slides</td><td>Electron Microscopy Sciences</td><td>63412-01</td><td></td></tr><tr><td>Microscope slides</td><td>Globe</td><td>1301</td><td></td></tr><tr><td>Liquid Blocker</td><td>Electron Microscopy Sciences</td><td>71312</td><td></td></tr><tr><td>VECTASHIELD mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>OCT medium</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Parafilm laboratory film</td><td>Fisher</td><td>13-374-10</td><td></td></tr><tr><td>Dow Corning high vacuum grease</td><td>Sigma-Aldrich</td><td>Z273554</td><td></td></tr><tr><td>Mouse anti-PSD95</td><td>Millipore</td><td>MABN68</td><td>antibody</td></tr><tr><td>Donkey anti-mouse Alexa 568</td><td>Invitrogen</td><td>A10037</td><td>antibody</td></tr><tr><td>Isolectin Alexa 488</td><td>Invitrogen</td><td>I21411</td><td></td></tr><tr><td>Chemiblocker</td><td>Chemicon</td><td>2170S</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Sulforhodamine</td><td>Sigma-Aldrich</td><td>341738</td><td></td></tr><tr><td>Carbodiimide</td><td>Thermo Scientific</td><td>22980</td><td>EDC</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr></tbody></table>

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.

Demonstrated here are two representative experiments that benefit from the use of a hydrophilized PTFE membrane to support wholemount retina during staining procedures. The first experiment, illustrates the method for fast and simple characterization of retinal vasculature, an elaborate network of blood vessels that span across multiple retinal layers (Figure 2). This approach combines bolus loading of isolectin with immersion labeling with SRH. This is an efficient method of labeling living tissue that ...

Watch this Scientific Journal Video about Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry at JoVE.com

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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 ...

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Research

JoVE Journal

Biology

23.0K Views.  Weill Medical College of Cornell University. 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.

Video: Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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. ...

Neuroscience

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

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

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

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.

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 ...

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 ...

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 ...

Synaptic Circuits Analysis and Protein Localization in the Mouse 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 characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

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

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

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is ...

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.

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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Chapters in this video

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

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

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

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

Preparation of Retinal Samples for Volume Electron Microscopy

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

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

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