We thank Carlos Mas, Mar&#237;a Pilar Crespo, and Cecilia Sampedro of CEMINCO (Centro de Micro y Nanoscop&#237;a C&#243;rdoba, CONICET-UNC, C&#243;rdoba, Argentina) for assistance in confocal microscopy, to Soledad Mir&#243; and Victoria Blanco for dedicated animal care and Laura Gatica for histological assistance. We also thank to Victor Diaz (Pro-Secretary of Institutional Communication of FCQ) for the video production and edition and Paul Hobson for his critical reading and language revision of the manuscript.
This article was funded by grants from Secretar&#237;a de Ciencia y Tecnolog&#237;a, Universidad Nacional de C&#243;rdoba (SECyT-UNC) Consolidar 2018-2021, Fondo para la Investigaci&#243;n Cient&#237;fica y Tecnol&#243;gica (FONCyT), Proyecto de Investigaci&#243;n en Ciencia y Tecnolog&#237;a (PICT) 2015 N&#176; 1314 (all to M.C.S.).

C57BL/6J mice were handled according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experimental procedures were designed and approved by the Institutional Animal Care and Use Committee (CICUAL) of the Faculty of Chemical Sciences, National University of C&#243;rdoba (Res. HCD 1216/18).
1. Preparation of buffer solutions and reagents
<ol>
	<li>Preparation of 1x phosphate buffer saline (PBS): Add 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 14.4 g of disodium-hydrogen-phosphate dihydrate (Na2HPO4 2H2O) and 0.24 g of potassium-dihydrogen phosphate (KH2PO4) to 800 mL of distilled water. Stir the solution until complete dissolution of the salts. Adjust the pH to 7.4 with chloride acid (HCl) and adjust the volume to 1 L with additional distilled water. Filter the solution and store it at 4 &#176;C.</li>
	<li>Preparation of 4% paraformaldehyde (PFA): Add 40 g of solid PFA to 800 mL of freshly prepared PBS. Stir the solution in a heating plate at a constant temperature of 60 &#176;C. Add 1 M sodium hydroxide (NaOH) until PFA is completely dissolved, checking that the pH is maintained at a range of 7.2-7.4. Top up the volume to 1,000 mL with PBS and mix. Turn off the heating plate and filter the solution when its temperature is under 35 &#176;C. Store the solution at 4 &#176;C for up to 3 days. 
	CAUTION: PFA is harmful if swallowed or if inhaled and is probably a mutagenic compound. Use gloves and face mask during the whole process and work in a safety cabin.</li>
	<li>Preparation of 1x Tris-buffered saline (TBS): Add 6.1 g of Tris and 9 g of NaCl to 800 mL of distilled water. Stir the solution until complete dissolution of the salts. Adjust pH to 7.4 with HCl and adjust the volume to 1 L with additional distilled water. Filter the solution and store it at 4 &#176;C.</li>
	<li>Preparation of TBS- 0.1% Triton X-100: Add 0.1 mL of TritonX-100 to 100 mL of TBS. Mix gently and store it at 4 &#176;C for up to 1 week. 
	NOTE: As Triton is a very dense detergent, slightly cut the terminal of the tip for better pipetting.</li>
	<li>Preparation of TBS-5%-Bovine Serum Albumin (BSA) -0.1% Triton-X-100: Weigh 5 g of BSA and add TBS- 0.1% triton X-100 solution up to a final volume of 100 mL. Stir gently. Aliquot and store at -20 &#176;C for up to 2 months.</li>
	<li>Isolectin GSA-IB4 Alexa fluor-488 conjugate (Isolectin GSA-IB4): Weight 36.75 mg of calcium chloride dihydrate (CaCl2&#183;2H2O) and add it to 500 mL of freshly prepared PBS. Stir the solution with a stir bar. Pipette 500 &#181;L of the solution of CaCl2/PBS and add it to the vial containing 500 &#181;g of lyophilized Isolectin GSA-IB4 powder. Mix gently and aliquot the solution in 0.5 mL tubes. Store them at -20 &#176;C protected from light.</li>
	<li>Poly(vinyl alcohol) in glycerol/Tris: Weigh 2.4 g of Poly(vinyl alcohol), add 6 g of glycerol and stir gently. Then, add 6 mL of water and continue stirring for several hours at room temperature. Add 12 mL of pre-warmed 0.2 M Tris solution (pH: 8.5) and heat at a constant temperature of 50 &#176;C for 10 min and mix occasionally. Finally, centrifuge the solution at 5,000 x g for 15 min for clarification. 
	&#8203;NOTE: Let the solution cool at room temperature, aliquot it and store it at -20 &#176;C.</li>
</ol>
2. Fluorescent lectin staining
<ol>
	<li>Upon mice sacrifice by carbon dioxide (CO2) inhalation, enucleate the eyes with scissors16. Fix the enucleated eyes with freshly prepared 4% PFA for 1 h at room temperature (RT). 
	NOTE: Fixation can also be performed by incubating the eyes in 4% PFA overnight (ON) at 4 &#176;C. Mice were sacrificed according to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Institutional Animal Care and Use Committee (CICUAL) of the Faculty of Chemical Sciences, National University of C&#243;rdoba (Res. HCD 1216/18).</li>
	<li>Under the dissecting microscope, remove the corneas with scissors by cutting along the limbus and dissect the whole retinas. Discard the anterior segment of the eye, and then separate the retina from the RPE-Choroid. 
	NOTE: Make sure to remove the remaining debris and hyaloid vessels out of the retina with forceps.</li>
	<li>Place the retinas in 200 &#181;L tubes. Then, block and permeabilize the retinas in 100 &#181;L of Tris-buffered saline (TBS) solution containing 5% Bovine Serum Albumin (BSA) and 0.1% Triton-X-100 during 6 h at 4 &#176;C with gentle agitation in the shaker. 
	NOTE: This step can be performed for 2 h at RT.</li>
	<li>Then, invert the tube to place the retina in the cap and remove the blocking solution with a pipette.</li>
	<li>Add 100 &#181;L of the solution containing 0.01 &#181;g/&#181;L of Isolectin GSA-IB4 (GSA-IB4). Wrap the tubes with aluminum foil to protect the samples from light. Incubate the retinas with the lectin solution ON at 4 &#176;C or for 2 h at RT with gentle agitation in the shaker.</li>
	<li>Wash the retina with 100 &#181;L of TBS containing 0.1% Triton-X-100 for 20 min with gentle agitation in the shaker. For proper washing, place the retina in the cap of the tube by inverting it. Return the tube to its standing position and remove the medium remaining in the container. Repeat this step three times.</li>
	<li>Place the retina in a slide and add a drop of PBS. Under the dissecting microscope, perform four equally distant cuts from the edges of the retina toward the optic nerve.</li>
	<li>To unfold the petals of the retina, use forceps or small pieces of filter paper. Ensure that the photoreceptor side is facing down.</li>
	<li>Remove the remaining PBS of the slide with filter paper. Then, add a mounting medium (Poly(vinyl alcohol) in glycerol/Tris) and coverslip. Let it dry for 1 h at RT.</li>
	<li>Store slides at 4 &#176;C protected from light. 
	&#8203;NOTE: Retinas can also be stored in PBS at 4 &#176;C until confocal microscopy visualization.</li>
</ol>
3. Confocal microscopy visualization and microphotograph acquisition
<ol>
	<li>Before visualization, incubate the slides for 10 min at RT covered from light.</li>
	<li>At the confocal microscope, place the slide in the plate face down.</li>
	<li>Focus the superior plexus of the retinal vasculature and select an area to start the image acquisition.</li>
	<li>Set general laser parameters in the software: Wavelength: 488 nm; Laser intensity; HV; Offset; Gain. 
	NOTE: Laser intensity, PMT, Offset, and gain may vary depending on the conditions and the usage of the lamp. The optimal settings provide a clear image of the vessels avoiding the saturation in the sensitivity of the photomultiplier tube to have a linear relationship with the fluorescence signal.</li>
	<li>Set other acquisition parameters: Mode; Size; Objective, without zoom; No Kalman; Step Size. 
	NOTE: Identical acquisition conditions must be used for imaging the control and experimental samples.</li>
	<li>Set the coordinates in x and y axis: Scan the retina in focus 2x. If the area shows vessels, select it by clicking twice the area in order to mark it for later image acquisition. 
	NOTE: It is highly recommendable to select retinal area by following the superior vascular plexus as the fluorescence of bigger vessels is higher.</li>
	<li>Move in the grid to a neighbor square, focus the retina, and select the area if it corresponds. Repeat this step until the selected area comprises the whole retina.</li>
	<li>Select one area and set the coordinates in z axis to define the thickness extent to scan. To do this, visualize the retina in focus 2x; with the micrometer, go to the top of the retina and click on Set at the Start position. Then, move to the bottom of the retina and click on Set at the End position.</li>
	<li>Repeat step 3.8 at every area selected in the grid. 
	NOTE: To verify the scanned area in depth, press Go at the Start position. If vessels are still visualized, readjust the position by moving the micrometer, and then click on Set. Repeat the process at the End position.</li>
	<li>Initialize automated scanning.</li>
	<li>Once the scanning process finishes, open the image.</li>
	<li>Select the Series format to record the image as a Mosaic and save it with the preferred extension. Finally, the stitched image will be shown on the screen.</li>
</ol>
4. Image processing
<ol>
	<li>In the ImageJ FIJI software, go to the Menu Bar and click on File &#62; Open. Select the image to process. In the Bio-Formats Import Options window, select the Display OME-XML metadata and click on OK. 
	NOTE: Images with similar name should be saved in separate folders.</li>
	<li>In the OME Metadata window, search for the pixel size information, named as PhysicalSizeX, PhysicalSizeY, and PhysicalSizeZ. Copy this data.</li>
	<li>For image calibration, go to the Menu Bar and select Image &#62; Properties. Copy the information of the image in step 4.2 and click on OK. The image opens as a window with several photos, where each one represents the microphotographs acquired at a z axis.</li>
	<li>Assign a preferred lut to the image in order to consign a color to the fluorescent label. To do that, go to the Menu Bar and click on the LUT icon.</li>
	<li>To visualize all stacks in a single image, go to the Menu Bar and select Image &#62; Stacks &#62; Z Project.</li>
	<li>In the Z Projection window displayed, select all the stacks where vessels are visualized. In Projection Type, choose Average Intensity and click on OK. 
	NOTE: The result of the sum of the stacks will be shown in a new image named AVG (image name).</li>
	<li>Adjust the brightness and contrast in the resulting image to reduce the background and highlight vessels. Go to the Menu Bar and click on Image&#62; Adjust&#62; Brightness/Contrast. In the B and C windows move the bars until a better intensity is observed. Click on Apply and save the changes.</li>
	<li>Copy the parameters selected for Brightness/Contrast; these must be identical for all images.</li>
	<li>Prior to image quantification, define the parameters that are going to be measured. In the Menu Bar, go to Analyze &#62; Set Measurements. In the procedures described here, it will be necessary to obtain Area and Perimeter (this is ultimately also needed to measure distances). Click on OK.</li>
	<li>Download the plugin required for vessel density quantification17&#160;and follow the installations instructions provided by the plugin.</li>
	<li>Continue with the quantification of a specific vascular parameter.</li>
</ol>
5. Quantification of avascular areas
<ol>
	<li>In the Menu Bar, choose the Wand Tool. Select the retinal area that lacks vessels. 
	NOTE: Bigger or smaller areas can be selected depending on the tolerance. To adjust the tolerance, click on the wand tool icon twice. Mode employed: Legacy.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 5.1 and 5.2 with all the avascular areas of the retina.</li>
	<li>Click on Measure in the ROI manager to obtain the avascular area information. The program will display a new window with the information required. Copy the data in another program or save the file. 
	NOTE: In some images, one may prefer to select the avascular areas manually. In this case, choose the Polygon Selection tool, draw short distances around the avascular area, and click on the image when a new distance is about to start. Close the selected area by clicking on the first point.</li>
	<li>Repeat steps 5.1, 5.2, and 5.3 to measure the total area of the retina.</li>
	<li>Calculate the avascular area of the retina as the sum of all the avascular areas measured divided by the total area of the retina.</li>
</ol>
6. Quantification of neovascular areas
<ol>
	<li>In the Menu Bar, choose the Wand tool.</li>
	<li>Zoom in the image to better visualize neovessels. Select the neovessels one by one with the wand tool. 
	NOTE: Adjust the tolerance no higher than 20 to ensure the selection of a single neovessel. This tolerance level must remain constant during all image quantifications.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 6.1 and 6.2 with all neovascular areas of the retina.</li>
	<li>Click on Measure in the ROI manager to obtain the area data. The program will display a new window with the information required. Copy the data in another program or save the file.</li>
	<li>Repeat steps 6.1, 6.2, and 6.3 to quantify the total area of the retina.</li>
	<li>Calculate the neovascular area of the retina as the sum of all the neovessels&#39; areas measured divided by the total area of the retina.</li>
</ol>
7. Quantification of vessel diameter
<ol>
	<li>In the Menu Bar, select the Straight-Line Tool.</li>
	<li>Zoom in the image to better visualize the vessel wall. Perform three transversal lines to the main vessel before the first branch. This must be done by drawing a line from the boundary of one wall of the vessel to the opposite. 
	NOTE: The line must be perfectly perpendicular to the wall vessel to avoid quantification errors.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 7.1 and 7.2 in all the vessels that need to be quantified.</li>
	<li>Click on Measure in the ROI manager to obtain the distance data. The program will display a new window with the information required. Copy the data in another program or save the file.</li>
</ol>
8. Quantification of vessel tortuosity
<ol>
	<li>In the Menu Bar, click on the Straight-Line Tool icon with the right button of the mouse and select Segmented Line or Freehand Line. Draw a line inside the vessel from the optic nerve until the first vessel ramification following&#160;the vascular shape. 
	NOTE: The line must follow the vascular shape to avoid quantification errors.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager.</li>
	<li>In the Menu Bar, select the Straight-Line Tool. Draw a line from the optic nerve until the first vessel ramification.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager.</li>
	<li>Click on Measure in the ROI Manager to obtain the distances data. The program will display a new window with the information required. Copy the data in another program or save the file.</li>
	<li>Calculate the tortuosity index as follows: distance obtained with Segmented line divided by the distance obtained with Straight line.</li>
</ol>
9. Quantification of vascular branching
<ol>
	<li>With the Oval Tool of the Menu Bar, define an area applied equally to all experimental conditions. The selected area must be a concentric circle drawn around the optic nerve.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. 
	NOTE: In this case, it is recommendable to save the ROI as the quantification is slow and the identical ROI must be used in all images. To do this, in the ROI Manager click on MORE &#62; Save.</li>
	<li>Manually, count the number of primary branches arising from main vessels inside the selection area.</li>
	<li>Repeat steps 9.2 and 9.3 in neighbor areas, maintaining the optic nerve-periphery criteria.</li>
</ol>
10. Quantification of vascular density
<ol>
	<li>Transform the image to 8-bit mode.</li>
	<li>Go to Plugins in the Menu Bar, select Vessel Analysis &#62; Vascular Density.</li>
	<li>Define an area with the Square tool of the Menu Bar, where the vessel density needs to be measured. Click on OK.</li>
	<li>The program will display a new window with the information required. Copy the data in another program or save the file.</li>
	<li>Repeat steps 10.2, 10.3, and 10.4 in the neighboring areas, maintaining the ROI but placing it nine times encompassing the periphery and the center of the whole retina.</li>
</ol>

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H., Wang, Z., Sun, Y., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ocular+angiogenesis:+from+development+to+pathologies.">Animal models of ocular angiogenesis: from development to pathologies.</a> FASEB Journal. 31 (11), 4665-4681 (2017).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Progress in Retinal and Eye Research. 29 (6), 500-519 (2010).</li><li>Kern, T. S., Antonetti, D. A., Smith, L. E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+diabetic+retinopathy:+Contribution+and+limitations+of+laboratory+research.">Pathophysiology of diabetic retinopathy: Contribution and limitations of laboratory research.</a> Ophthalmic Research. 62 (4), 196-202 (2019).</li><li>Lorenc, V. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1R+regulates+the+extracellular+level+of+active+MMP-2,+pathological+neovascularization,+and+functionality+in+retinas+of+OIR+mouse+model.">IGF-1R regulates the extracellular level of active MMP-2, pathological neovascularization, and functionality in retinas of OIR mouse model.</a> Molecular Neurobiology. 55 (2), 1123-1135 (2018).</li><li>Ma, N., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+microglia+as+passenger+leukocytes+to+the+fate+of+intraocular+neuronal+retinal+grafts.">Contribution of microglia as passenger leukocytes to the fate of intraocular neuronal retinal grafts.</a> Investigative Ophthalmology &amp; Visual Science. 39 (12), 2384-2393 (1998).</li><li>Mazzaferri, J., Larriv&#233;e, B., Cakir, B., Sapieha, P., Costantino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+machine+learning+approach+for+automated+assessment+of+retinal+vasculature+in+the+oxygen+induced+retinopathy+model.">A machine learning approach for automated assessment of retinal vasculature in the oxygen induced retinopathy model.</a> Scientific Reports. 8 (1), 3916 (2018).</li><li>Milde, F., Lauw, S., Koumoutsakos, P., Iruela-Arispe, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+retina+in+3D:+quantification+of+vascular+growth+and+remodeling.">The mouse retina in 3D: quantification of vascular growth and remodeling.</a> Integrative Biology: Quantitative Biosciences from Nano to Macro (Camb). 5 (12), 1426-1438 (2013).</li><li>Yang, 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=Pericytes+of+indirect+contact+coculture+decrease+integrity+of+inner+blood-retina+barrier+model+in+vitro+by+upgrading+MMP-2/9+activity.">Pericytes of indirect contact coculture decrease integrity of inner blood-retina barrier model in vitro by upgrading MMP-2/9 activity.</a> Disease Markers. 2021, 7124835 (2021).</li><li>Huang, Q., Wang, S., Sorenson, C. M., Sheibani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF-deficient+mice+exhibit+an+enhanced+rate+of+retinal+vascular+expansion+and+are+more+sensitive+to+hyperoxia-mediated+vessel+obliteration.">PEDF-deficient mice exhibit an enhanced rate of retinal vascular expansion and are more sensitive to hyperoxia-mediated vessel obliteration.</a> Experimental Eye Research. 87 (3), 226-241 (2008).</li><li>Jiang, H., Zhang, H., Jiang, X., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+D-amino+acid+oxidase+prevents+retinal+neurovascular+pathologies+in+diabetic+rats.">Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.</a> Diabetologia. 64 (3), 693-706 (2021).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Animal models of retinopathies are powerful tools for studying vascular development, remodeling, or pathological angiogenesis. The success of these studies in the field relies on the easy access to the tissue that allows to perform a wide variety of techniques, providing data from in vivo and postmortem mice26,27. Moreover, great correlation has been found between in vivo studies and clinical analysis, providing solid traceability and r...

The eyes are nourished by two arterio-venous system: the choroidal vasculature, an external vascular network that irrigates retinal pigmented epithelium and photoreceptors; and the neuro-retinal vasculature that irrigate the ganglion cells layer and the inner nuclear layer of the retina1. The retinal vasculature is an organized network of vessels that deliver nutrients and oxygen to the retinal cells and harvest waste products to ensure proper visual signaling transduction. This vasculature has some distinct features, including: the lack of autonomous innervation, the regulation of vascular tone by intrinsic retinal mechanisms and the possession of a complex retinal-blood barrier2. Therefore, retinal vasculature has been the focus of many researchers who have extensively studied not only vasculogenesis during the development, but also the alterations and the pathological angiogenesis that these vessels undergo in diseases3. The most common vascular changes observed in retinopathies are vessel dilatation, neovascularization, loss of vascular arborization and deformation of the retinal main vessels, which makes them more ziggaggy4,5,6. One or more of the described alterations are the earliest signs to be detected by clinicians. Vascular visualization provides a rapid, non-invasive, and inexpensive screening method7. The extensive study of the alterations observed in the vascular tree will determine whether the retinopathy is non-proliferative or proliferative and the further treatment. The non-proliferative retinopathies can manifest themselves with aberrant vascular morphology, decreased vascular density, acellular capillaries, pericytes death, macular edema, among others. In addition, proliferative retinopathies also develop increased vascular permeability, extracellular remodeling, and the formation of vascular tufts toward the vitreous cavity that easily breakdown or induce retinal detachment8.
Once detected, the retinopathy can be monitored through its vascular changes9,10. The progression of the pathology can be followed through the structural changes of the vessels, which clearly define stages of the disease11. The quantification of vascular alterations in these models allowed to correlate vessel changes and neuronal death and to test pharmacological therapies for patients in different phases of the disease.
In light of the above statements, we consider that the recognition and quantification of vascular alterations are fundamental in retinopathies studies. In this work, we will show how to measure different vascular parameters. To do that, we will employ two animal models. One of them is the Oxygen-induced retinopathy mouse model12, which mimics Retinopathy of Prematurity and some aspects of proliferative Diabetic Retinopathy13,14. In this model, we will measure avascular areas, neovascular areas and the dilatation and tortuosity of main vessels. In our laboratory, a Metabolic Syndrome (MetS) mouse model has been developed, which induces a non-proliferative retinopathy15. Here, we will evaluate vascular density and branching.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminuim foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck</td>
			<td>A4503</td>
			<td>quality</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Biopack</td>
			<td>9632.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope FV1200</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td>with motorized plate</td>
		</tr>
		<tr>
			<td>Covers</td>
			<td>Paul Marienfeld GmnH &#38; Co.</td>
			<td>111520</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>NIKON</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium-hydrogen-phosphate dihydrate</td>
			<td>Merck</td>
			<td>119753</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L&#160; tube</td>
			<td>Merck</td>
			<td>Z316121</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merck</td>
			<td>WHA5201090</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker GyroMini</td>
			<td>LabNet International</td>
			<td>S0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 Conjugate</td>
			<td>Invitrogen</td>
			<td>I21411</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol) (Mowiol 4-88)</td>
			<td>Merck</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>pHmeter</td>
			<td>SANXIN</td>
			<td>PHS-3D-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-dihydrogen phosphate</td>
			<td>Merck</td>
			<td>1,04,873</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Merck</td>
			<td>GE17-1321-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>X100-1GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Analysis Fiji software</td>
			<td>Mai Elfarnawany</td>
			<td></td>
			<td>https://imagej.net/Vessel_Analysis</td>
		</tr>
	</tbody>
</table>

quantification of vascular parameters in whole mount retinas of mice with non-proliferative and proliferative retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

As described in the protocol section, from a single fluorescent staining assay you can obtain the vascular morphology and evaluate several parameters of interest quantitatively. The search of a specific alteration will depend on the type of retinopathy studied. In this article, avascular and neovascular areas, tortuosity, and dilatation were evaluated in a mouse model of proliferative retinopathy, whereas the vascular branching and density were analyzed in a MetS mouse model, which induces a non-proliferative retinopathy...

Watch this Scientific Journal Video about Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies at JoVE.com

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

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3.4K Views.  Universidad Nacional de Córdoba. This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Video: Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...