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. 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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 ...

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