Name: retinal cryo-sections, whole-mounts, and hypotonic isolated vasculature preparations for immunohistochemical visualization of microvascular pericytes
Uploaded: 2018-10-07T14:00:00
Description: Watch this Scientific Journal Video about Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes at JoVE.

The research was funded by The Lundbeck Foundation, Denmark.

The protocol was optimized and demonstrated on adult male albino rats. 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. Animals were euthanized by carbon dioxide and subsequent cervical dislocation.
1. Rat Retinal Tissue Preparations
<ol>
	<li>Cryo-section
	<ol>
		<li>Make posterior and anterior ~0.5 cm slits of in the rat eyelid with a scalpel.
		<ol>
			<li>Opti...

<ol>
	<li>Eshaq, R. S., Aldalati, A. M. Z., Alexander, J. S., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy:+Breaking+the+barrier.">Diabetic retinopathy: Breaking the barrier.</a> Pathophysiology. , (2017).</li><li>Cai, W., 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+in+Brain+Injury+and+Repair+After+Ischemic+Stroke.">Pericytes in Brain Injury and Repair After Ischemic Stroke.</a> Translational Stroke Research. , (2016).</li><li>Trost, A., 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=Brain+and+Retinal+Pericytes:+Origin,+Function+and+Role.">Brain and Retinal Pericytes: Origin, Function and Role.</a> Frontiers in Cellular Neuroscience. 10, 20 (2016).</li><li>Ramos, D., Lagali, N., 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=The+Use+of+Confocal+Laser+Microscopy+to+Analyze+Mouse+Retinal+Blood+Vessels.">The Use of Confocal Laser Microscopy to Analyze Mouse Retinal Blood Vessels.</a> Confocal Laser Microscopy - Principles and Applications in Medicine, Biology, and the Food Sciences. , (2013).</li><li>Moran, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+cross+talk+in+diabetic+retinopathy:+Pathophysiological+roles+and+therapeutic+implications.">Neurovascular cross talk in diabetic retinopathy: Pathophysiological roles and therapeutic implications.</a> American Journal of Physiology. Heart and Circulatory Physiolog. 311, H738-H749 (2016).</li><li>Henkind, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculation+of+the+peripapillary+retina.">Microcirculation of the peripapillary retina.</a> Transactions - American Academy of Ophthalmology and Otolaryngology. 73, 890-897 (1969).</li><li>Attwell, D., Mishra, A., Hall, C. N., O'Farrell, F. M., Dalkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+a+pericyte?.">What is a pericyte?.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 36, 451-455 (2016).</li><li>Fernandez-Bueno, I., 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=Histologic+Characterization+of+Retina+Neuroglia+Modifications+in+Diabetic+Zucker+Diabetic+Fatty+Rats.">Histologic Characterization of Retina Neuroglia Modifications in Diabetic Zucker Diabetic Fatty Rats.</a> Investigative Ophthalmology &amp; Visual Science. 58, 4925-4933 (2017).</li><li>Yu, D. -. Y., Yu, P. K., Cringle, S. J., Kang, M. H., Su, E. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+characteristics+of+the+retinal+and+choroidal+vasculature.">Functional and morphological characteristics of the retinal and choroidal vasculature.</a> Progress in Retinal and Eye Research. 40, 53-93 (2014).</li><li>Allen, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severity+of+middle+cerebral+artery+occlusion+determines+retinal+deficits+in+rats.">Severity of middle cerebral artery occlusion determines retinal deficits in rats.</a> Experimental Neurology. 254, 206-215 (2014).</li><li>Kyhn, M. V., 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=Acute+retinal+ischemia+caused+by+controlled+low+ocular+perfusion+pressure+in+a+porcine+model.+Electrophysiological+and+histological+characterisation.">Acute retinal ischemia caused by controlled low ocular perfusion pressure in a porcine model. Electrophysiological and histological characterisation.</a> Experimental Eye Research. 88, 1100-1106 (2009).</li><li>Blixt, F. W., Radziwon-Balicka, A., Edvinsson, L., Warfvinge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+CGRP+and+its+receptor+components+CLR+and+RAMP1+in+the+rat+retina.">Distribution of CGRP and its receptor components CLR and RAMP1 in the rat retina.</a> Experimental Eye Research. 161, 124-131 (2017).</li><li>Sarlos, S., Wilkinson-Berka, J. 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+renin-angiotensin+system+and+the+developing+retinal+vasculature.">The renin-angiotensin system and the developing retinal vasculature.</a> Investigative Ophthalmology &amp; Visual Science. 46, 1069-1077 (2005).</li><li>Wittig, D., Jaszai, J., Corbeil, D., Funk, R. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+and+characterization+of+putative+mesenchymal+stem+cell+markers+in+the+retinal+capillary+network+of+rodents.">Immunohistochemical localization and characterization of putative mesenchymal stem cell markers in the retinal capillary network of rodents.</a> Cells Tissues Organs. 197, 344-359 (2013).</li><li>Tual-Chalot, S., Allinson, K. R., Fruttiger, M., Arthur, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+immunofluorescent+staining+of+the+neonatal+mouse+retina+to+investigate+angiogenesis+in+vivo.">Whole mount immunofluorescent staining of the neonatal mouse retina to investigate angiogenesis in vivo.</a> Journal of Visualized Experiments. , e50546 (2013).</li><li>Park, D. Y., 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=Plastic+roles+of+pericytes+in+the+blood-retinal+barrier.">Plastic roles of pericytes in the blood-retinal barrier.</a> Nature Communications. 8, 15296 (2017).</li><li>Hughes, S., Chan-Ling, T. <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+smooth+muscle+cell+and+pericyte+differentiation+in+the+rat+retina+in+vivo.">Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.</a> Investigative Ophthalmology &amp; Visual Science. 45, 2795-2806 (2004).</li><li>Lange, 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=Intravitreal+injection+of+the+heparin+analog+5-amino-2-naphthalenesulfonate+reduces+retinal+neovascularization+in+mice.">Intravitreal injection of the heparin analog 5-amino-2-naphthalenesulfonate reduces retinal neovascularization in mice.</a> Experimental Eye Research. 85, 323-327 (2007).</li><li>Higgins, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diltiazem+reduces+retinal+neovascularization+in+a+mouse+model+of+oxygen+induced+retinopathy.">Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy.</a> Current Eye Research. 18, 20-27 (1999).</li><li>Chou, J. C., Rollins, S. D., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+digest+protocol+to+analyze+the+retinal+vasculature+of+a+mouse+model.">Trypsin digest protocol to analyze the retinal vasculature of a mouse model.</a> Journal of Visualized Experiments. , e50489 (2013).</li><li>Hazra, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+X+receptor+modulates+diabetic+retinopathy+outcome+in+a+mouse+model+of+streptozotocin-induced+diabetes.">Liver X receptor modulates diabetic retinopathy outcome in a mouse model of streptozotocin-induced diabetes.</a> Diabetes. 61, 3270-3279 (2012).</li><li>Zhang, L., Xia, H., Han, Q., Chen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+antioxidant+gene+therapy+on+the+development+of+diabetic+retinopathy+and+the+metabolic+memory+phenomenon.">Effects of antioxidant gene therapy on the development of diabetic retinopathy and the metabolic memory phenomenon.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 253, 249-259 (2015).</li><li>Dagher, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+rat+and+human+retinas+predict+a+role+for+the+polyol+pathway+in+human+diabetic+retinopathy.">Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.</a> Diabetes. 53, 2404-2411 (2004).</li><li>Navaratna, D., McGuire, P. G., Menicucci, G., Das, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+degradation+of+VE-cadherin+alters+the+blood-retinal+barrier+in+diabetes.">Proteolytic degradation of VE-cadherin alters the blood-retinal barrier in diabetes.</a> Diabetes. 56, 2380-2387 (2007).</li><li>Gustavsson, 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=Vascular+cellular+adhesion+molecule-1+(VCAM-1)+expression+in+mice+retinal+vessels+is+affected+by+both+hyperglycemia+and+hyperlipidemia.">Vascular cellular adhesion molecule-1 (VCAM-1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia.</a> PLoS One. 5, e12699 (2010).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+blood+flow+in+the+retinal+trilaminar+vascular+network.">Regulation of blood flow in the retinal trilaminar vascular network.</a> Journal of Neuroscience. 34, 11504-11513 (2014).</li><li>Puro, 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=Retinovascular+physiology+and+pathophysiology:+new+experimental+approach/new+insights.">Retinovascular physiology and pathophysiology: new experimental approach/new insights.</a> Progress in Retinal and Eye Research. 31, 258-270 (2012).</li></ol>

We present three retinal preparation techniques that can be applied in the study of microvascular retinal pericytes. Below, we provide a comparison between each of the methods and highlight critical steps in the protocols.
With cryo-sectioning, the retina is cut in sagittal sections and hence, it is possible to obtain numerous specimens from the same retina. The numeral sections resulting from this method make it an ideal choice for antibody specificity and titration testing as it prevents unn...

Retinal pericytes are the focus of many research laboratories as these cells play a major role in the integrity of the vasculature. Pathological conditions such as diabetic retinopathy1, ischemia2, and glaucoma3 have vascular characteristics that involve the function of pericytes. Pericytes are found in the inner retinal capillary plexuses. The central retinal artery that supplies the inner retina branches into two layers of capillary plexuses. The inner vascular bed is situated between the ganglion cell and inner nuclear layers. The deeper layer is more dense and complex and is localized between the inner and outer nuclear layers4,5. In addition, some parts of the retina also contain a third network termed the radial parapapillary capillaries. These are long, straight capillaries that lie among the nerve fibers and rarely anastomose with one another or the other two plexuses6. Within the capillary wall, pericytes are embedded in the basement membrane and line the abluminal side of vascular endothelial cells.
To this date, there is no unique biological marker of these pericytes that can differentiate them from other vascular cells. Platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2) are commonly used markers which both present on pericytes but also other vascular cells. Identification of pericytes is further complicated by the existence of pericyte subsets that vary in morphology and protein expression7. Currently, the best identification relies on a combination of protein markers and the characteristic positioning of the pericyte in the vascular wall. We demonstrate here three different tissue preparation techniques for immunohistochemical PDGFR&#946;/NG2 staining of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.
With cryo-sections, the retina and sclera are cut through the optic nerve. This allows for the visualization of all layered structures of neurons. The distinct ten layers of the retina are apparent as interchanging nuclear and axonal/dendritic structures that can be visualized with stains such as hematoxylin/eosin or fluorescent nuclear 4',6-diamidino-2-phenylindole (DAPI)8. The metabolic requirements differ between the layers9 and it provides a method to determine the thickness or total absence of a specific layer (e.g., the loss of retinal ganglion cells is one of the hallmarks of retinal ischemia10,11). The vasculature is evident as transverse cuts through the retina, making it possible to separately study the capillary plexuses within the respective retinal layers12,13.
More traditionally, the investigations of the retinal vasculature network are performed in retinal whole-mounts. With this tissue preparation, the retina is cut and flattened as a flower-shaped structure. The method is a relatively fast tissue preparation technique that can highlight the overall architecture retinal vasculature and it is therefore often applied in the investigation of neovascularization in the murine retina. Successful visualization of the microvasculature in whole-mounted retinas is also reported in the developing neonatal mouse and rat retina14,15,16,17,18,19. These studies reveal a more defined pericytic activity with larger capillary-free areas in the adult compared to the neonatal retina14.
Another way of visualizing is the retinal microvasculature after hypotonic isolation. This tissue preparation technique results in retinal blood vessels and capillaries being freed of the neuronal cells. This type of two-dimensional imaging of the isolated retinal vascular network is usually performed after retinal trypsin digestion20 and used to assess the vascular abnormalities of diabetic retinopathy including pericyte loss and capillary degeneration20,21,22. The hypotonic isolation method offers the investigations of retinal vascular gene and protein regulatory responses as they has been done with RT-PCR and western blotting23,24,25. We provide here a protocol for the free-float immunohistochemical staining of the hypotonic isolated retinal vasculature as an alternative to trypsin digestion to examine the microvascular pericytes.

<table border="0" cellpadding="0" cellspacing="0" width="902">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Geletin from porcine skin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">G2625-500G</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Albumin from chicken egg white</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">A5253-500G</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Deoxyribonuclease (DNAse) I from bovine pancreas</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">D5025-15KU</td>
			<td style="width:171px;">Dissolved in 0.15 M NaCl</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Bovine serum albumin (BSA)</td>
			<td style="width:175px;">VWR</td>
			<td style="width:165px;">0332-100G</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Normal donkey serum</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">017-000-121, lot 129348</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Rabbit anti-PDGFR&#946;</td>
			<td style="width:175px;">Santa Cruz</td>
			<td style="width:165px;">sc-432</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Mouse anti-NG2</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:165px;">ab50009</td>
			<td style="width:171px;">1:500</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">711-585-152</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">715-095-151</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Cy2 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">711-225-152</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Cy3 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">715-165-150</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">D9542-1MG</td>
			<td style="width:171px;">Dissolved in DMSO</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Anti-fading mounting medium</td>
			<td style="width:175px;">Vector Laboratories</td>
			<td style="width:165px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Anti-fading mounting medium with DAPI</td>
			<td style="width:175px;">Vector Laboratories</td>
			<td style="width:165px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Nunc Lab-Tek II 4-well chamber slide</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:165px;">154526</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The successful protocols provide three different retinal preparations for visualizing microvascular pericytes. Each of these methods uses the PDGFR&#946; and NG2 immunoreactivity co-localization and the unique position of the pericytes that wrap around the capillary endothelium foridentification.
With cryo-sections, the neuronal layers can be identified by the fluorescent density of DAPI-labelled nuclei and the inner and deep ca...

Watch this Scientific Journal Video about Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes at JoVE.

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

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 ...

These methods can help answer key questions in the ophthalmology field such as pericyte proliferation or migration. The main advantage of these procedure is that they provide a variety of visualization options. Demonstrating the procedures will be Karin Dreisig and Frank W.Blixt, two post-docs at our laboratory.To enucleate the eye, first use a scalpel to make the posterior and anterior slits of approximately 0.5 centimeters in the rat eyelid. Grab the eye with forceps and tilt carefully to the side to expose the surrounding tissue. Enucleate the eye by making cuts with dissection scissors in the connective and muscular tissue.Briefly place the eye in 4%formaldehyde and phosphate-buffered saline for one to two minutes. Rinse briefly in sodium buffer before making a hole at the corneal limbus by applying light pressure with the tip of a scalpel. Cut along the corneal limbus with dissection scissors to remove the cornea and lens with forceps.Submerge the remaining tissue with retina in 4%formaldehyde in PBS for two to four hours. Cryo-protect the tissue by immersing in Sorenson's phosphate buffer containing 10%sucrose and then 25%sucrose. Transfer the tissue once it has sunk to the bottom of the container.Embed in Yazulla medium prepared with 3%gelatine from porcine skin and 30%albumin from chicken egg white. Next, cut 10-micron vertical cryosections of the gelatin-embedded retina through to the optic nerve. Place the cryosections on a glass slide and allow to dry for a minimum of one hour.Once dry, submerge the slide in PBS with 0.25%Triton-X 100 for 15 minutes. Then, drip a mixture of 1:100 PDGF receptor beta and 1:500 NG2 primary antibodies in PBST and 1%BSA onto the cryosection and place in a moist chamber at four degrees Celsius overnight. The next day, wash the sections by immersing the slide in PBST twice for 15 minutes each time.Then drip a mixture of 1:100 Anti-Mouse Alexa Fluor 594-linked and 1:100 Anti-Rabbit FITC-linked secondary antibodies diluted in PBST with 3%BSA onto the cryosections and incubate for one hour in the dark. After the incubation, rinse the glass slide in PBST twice for 15 minutes each time. Mount the stained cryosections with anti-fading mounting medium containing DAPI and a coverslip.A second retinal tissue preparational technique is whole-mount. Following the removal of the cornea, use small opening movements of forceps to separate the retina from the retinal pigment epithelium towards the optic nerve. Free the retina at the optic nerve with dissection scissors and make three to four slits of a few millimeters'length from the retinal periphery towards the optic nerve head.Spread the retina onto a glass slide and allow to dry for five to 10 minutes. Drip 4%formaldehyde onto the retina and fix for 20 to 30 minutes. Following fixation, rinse with PBS.For optimal results, immunostain directly after rinsing. First, drip PBST onto the whole-mount and incubate at room temperature for 15 minutes. Pour off the PBST, then add the primary antibody solution and incubate in a moist chamber at four degrees Celsius overnight.The next day, pour off the primary antibody and drip on PBST to rinse the retina twice for 15 minutes each time. After pouring off the second wash, drip secondary antibody solution onto the retina and incubate for one hour in a moist chamber at room temperature in the dark. Following the secondary antibody incubation, wash twice with PBST as before.Mount the stained whole-mount with anti-fading mounting medium containing DAPI and a cover slip. Alternative to whole-mounting the retina, the retinal vasculature can be isolated by hypotonic isolation. First, place the retina in one milliliter of deionized water in a 24-well plate and shake at 200 rpm with a 1.5-millimeter vibration orbit for one hour at room temperature, then add 200 units of DNAse 1 to dissociate the lysed cell debris from the retinal vasculature and shake for another 30 minutes at room temperature.Rinse the retina in deionized water for three times for five minutes each with shaking at 150 to 300 rpm to remove neuronal cell debris. The retina should become more transparent with each rinse, indicative of the removal of neuronal cellular debris. After rinsing, fix the retina in one milliliter of 4%paraformaldehyde and PBS for 10 minutes at room temperature.Rinse with PBS three times. If proceeding to immunostaining, block the hypotonic-isolated vasculature with 500 microliters of 10%donkey serum diluted in PBS for one hour with shaking at 100 rpm. After blocking, incubate the retina overnight in 600 microliters of primary antibody solution consisting of 1:100 PDGF-receptor beta and 1:500 NG2 primary antibodies diluted in 10%donkey serum and PBS.The next day, rinse the retinal network three times in PBS for five minutes each time, then incubate in 1:100 Anti-Mouse Alexa Fluor 594th-linked and 1:100 Anti-Rabbit FITC-linked secondary antibodies, diluted in 10%donkey serum and PBS with shaking at 100 rpm at room temperature for one hour in the dark. Following a five-minute wash in PBST, incubate in 0.2 nanograms-per-milliliter DAPI and PBST for 15 minutes. Wash three times for five minutes with PBST while protected from light.Next, cut the tip of a plastic Pasteur pipette. Remove sharp edges using a pipette tip, and moisten the Pasteur pipette with PBST, then aspirate the retinal vasculature into the pipette and dispense into a four-well glass chamber slide. The moistening step is important to avoid the retinal vasculature sticking to the inside of the Pasteur pipette.Position the retinal vasculature by tilting the chamber slide back and forth. Remove the medium from the wells of the chamber slide. The surface tension of the liquid will flatten the vasculature onto the bottom of the slide.If needed, drip drops of liquid onto the retinal vasculature to unfold. Ensure correct unfolding under a microscope before removing the plastic wells from the chamber slide. Mount the stained vasculature with anti-fading mounting medium and a cover slip.NG2 immunohistochemistry reveals positive vessels within the inner part of the retina. Arrows indicate circular and horseshoe-shaped immunoreactivity in the vessels. The arrowhead points out a longitudinally-cut vessel.PDGF-receptor beta immunohistochemistry shows similar immunoreactivity to NG2. Again the arrows and arrowhead indicate immunoreactivity in the vessels. This image shows a merge of NG2 in green, PDGF-receptor beta in red, and DAPI in blue.By superimposing the three different filters, it is revealed that NG2 and PDGF-receptor beta are co-localized. This image shows an immunostained whole-mount preparation with NG2 staining in red. Immunoreactivity is visible along the superficial vascular network with intense staining in abluminal pericytes.The neuronal cells are visible as DAPI blue nuclei between the NG2-stained microvasculature. This image shows the hypotonic isolated rat retinal vascular network stained with NG2 in green and PDGF-receptor beta in red. The complete network showed NG2-immunoreactivity.PDGF-receptor beta immunoreactivity was found in cell somas. At higher magnification, it is clear that PDGF-receptor beta staining is only seen in cell somas in the vascular network, indicating pericyte immunoreactivity. Including DAPI staining together with NG2 and PDGF-receptor beta reveals three pericytes in two undefined vessel wall cells in this high-magnification image.Following these procedures, other immunostainings like smooth muscle cell or endothelial stainings can be used in order to answer additional questions. For example, how do cells in the retinal vasculature look following ischemia?

retinal-cryo-sections-whole-mounts-hypotonic-isolated-vasculature

Research

JoVE Journal

Biology

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...