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>
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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?

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Research

JoVE Journal

Biology

9.9K visualizações.  Glostrup Research Institute. Esses métodos podem ajudar a responder a perguntas-chave no campo da oftalmologia, como a proliferação de pericílitos ou a migração. A principal vantagem deste procedimento é que eles fornecem uma variedade de opções de visualização. Demonstrando os procedimentos serão Karin Dreisig e Frank W.Blixt, dois pós-doutores em nosso laboratório.Para enuclear o olho, primeiro use um bisturi para fazer as fendas posteriores e anteriores de aproximadamente 0,5 centímetros na pálp...

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