Name: Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes (Video) | JoVE Journal
Uploaded: 2018-10-07T14:00:00.000+00:00
Duration: 10 min 46 s
Description: Learn three retinal tissue preparation techniques for immunohistochemical visualization of rat microvascular pericytes. Cryo-sections, whole-mounts, and hypotonic isolation enable detailed study of pericyte dynamics in ophthalmology research.

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5 minutes

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>Optional: Using a diathermy burner, mark the eye at the inner angle to orientate the eye in a uniform fashion during embedding and allow for vertical cryostat sectioning through the optic nerve.</li>
		</ol>
		</li>
		<li>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. 
		CAUTION: Do not pull the eye too hard as excessive pressure on the optic nerve may cause retinal detachment.</li>
		<li>Put the eye briefly in 4% formaldehyde in phosphate-buffered saline (PBS) to stabilize before making an initial hole at the corneal limbus by applying light pressure with the tip of a scalpel.</li>
		<li>Under a microscope, cut along the corneal limbus with dissection scissors to remove the cornea and remove the lens with forceps before submerging in 4% formaldehyde in PBS for 2&#8211;4 h.</li>
		<li>Rinse sequentially in S&#246;rensen&#39;s phosphate buffer with 10% sucrose and 25% sucrose.</li>
		<li>Embed in Yazulla medium prepared with 3% gelatin from porcine skin and 30% albumin from chicken egg white. 
		NOTE: The protocol can be paused here with tissue storage at -20&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Whole-mount
	<ol>
		<li>Make posterior and anterior ~0.5 cm slits in the rat eyelid with a scalpel.</li>
		<li>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. 
		CAUTION: Do not pull the eye too hard as excessive pressure on the optic nerve may cause retinal detachment.</li>
		<li>Put the eye briefly in 4% formaldehyde in PBS to stabilize before making an initial hole at the corneal limbus by applying light pressure with the tip of a scalpel.</li>
		<li>Under a microscope, cut along the corneal limbus with dissection scissors to remove the cornea and remove the lens with forceps.</li>
		<li>Separate the retina from the retinal pigment epithelium towards the optic nerve with forceps using small opening movements to avoid major tearing.</li>
		<li>Free the retina at the optic nerve with dissection scissors and make four slits of a few millimeters length from the retinal periphery towards the optic nerve head.</li>
		<li>Spread the retina onto a glass slide and allow drying for 5&#8211;10 min.</li>
		<li>Fix in 4% formaldehyde for 20&#8211;30 min by dripping formaldehyde onto the retina. 
		CAUTION: Do not apply directly on the retina as it may detach from the glass.</li>
		<li>Rinse with PBS. For optimal results, immuno-stain directly after rinsing.</li>
	</ol>
	</li>
	<li>Hypotonic isolation
	<ol>
		<li>Make posterior and anterior ~0.5 cm slits in the rat eyelid with a scalpel.</li>
		<li>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. 
		CAUTION: Do not pull the eye too hard as excessive pressure on the optic nerve may cause retinal detachment.</li>
		<li>Make an initial hole at the corneal limbus by applying light pressure with the tip of a scalpel.</li>
		<li>Under a microscope, cut along the corneal limbus with dissection scissors to remove the cornea and remove the lens with forceps.</li>
		<li>Separate the retina from the retinal pigment epithelium towards the optic nerve head with forceps using small opening movements to avoid major tearing.</li>
		<li>Free the retina at the optic nerve with dissection scissors, place the retina in 1 mL of deionized water in a 24-well plate and shake at 200 rpm with a 1.5 mm vibration orbit for 1 h at room temperature. 
		NOTE: Hereafter, the retina will appear less defined at the edges.</li>
		<li>Add 200 U DNAse 1 to dissociate the lysed cell debris from the retinal vasculature and shake for another 30 min at room temperature. 
		NOTE: Debris might start to form in the wells.</li>
		<li>Rinse minimum 3 times in deionized water for 5 min with shaking at 150&#8211;300 rpm to remove neuronal cell debris. The retina should become more transparent with each rinse indicative of the removal of neuronal cellular debris.
		<ol>
			<li>Use a dark background to look into the 24-well plate to clearly see the diaphanous isolated retinal vasculature.</li>
			<li>(Optional):&#160;If the vasculature does not appear free of neuronal layers (semi-transparent) at this point either add more rinse steps, increase the shaking speed or use a pipet to aspirate liquid onto the vasculature. 
			CAUTION: Either of the optional steps may damage the vasculature.</li>
		</ol>
		</li>
		<li>Fix 10 min in 1 mL of 4% paraformaldehyde in PBS at room temperature and rinse 3 times in PBS. 
		NOTE: The protocol can be paused here with tissue storage at 4&#160;&#176;C.</li>
	</ol>
	</li>
</ol>
2. Immunohistochemistry
<ol>
	<li>Staining of cryo-sections
	<ol>
		<li>Cut 10 &#181;m cryo-sections of the gelatin-embedded retina as vertical sectioning through the optic nerve and place the cryo-sections on a glass slide and let dry (minimum 1 h).</li>
		<li>Submerge the glass slide in PBS with 0.25 % Triton X-100 (PBS-T) for 15 min.</li>
		<li>Drip 1:100 PDGFR&#946; and 1:500 NG2 primary antibodies diluted in PBS-T + 1% BSA onto the cryo-section and incubate in incubation chambers at 4&#160;&#176;C overnight.</li>
		<li>Submerge the glass slide 2 times in PBS-T for 15 min and drip 1:100 anti-mouse Alexa Fluor 594-linked and 1:100 anti-rabbit FITC-linked secondary antibodies diluted in PBS-T with 3% BSA onto the cryo-sections.</li>
		<li>Incubate the glass slide 1 h at room temperature in the dark.</li>
		<li>Rinse the glass slide in PBS-T 2x 15 min. 
		NOTE: Optional: For double and triple immunofluorescent staining, sequential staining can be performed by repeating the procedure from 2.1.3 to 2.1.6 two and three times, respectively.</li>
		<li>Mount the stained cryo-sections with anti-fading mounting medium containing DAPI and a coverslip.</li>
	</ol>
	</li>
	<li>Staining of whole-mount
	<ol>
		<li>Drip PBS-T onto the whole-mount and incubate at room temperature for 15 min.</li>
		<li>Pour off and drip 1:100 PDGFR&#946; and 1:500 NG2 primary antibodies diluted in PBS-T + 1% BSA and incubate in moist chambers at 4&#160;&#176;C overnight.</li>
		<li>Pour off and drip on PBS-T to rinse the glass slide in 2x 15 min.</li>
		<li>Pour off and drip on 1:100 anti-rabbit Cy2- and 1:100 anti-mouse Cy3-linked secondary antibody diluted in PBS-T with 3% BSA to incubate 1 h in a moist chamber at room temperature in the dark.</li>
		<li>Pour off and drip on PBS-T to rinse in 2x 15 min in the dark. 
		NOTE: Optional: For double and triple immunofluorescent staining, sequential staining can be performed by repeating the procedure from 2.2.2 to 2.2.5 two and three times, respectively.</li>
		<li>Mount the stained whole-mount with anti-fading mounting medium containing DAPI and a coverslip.</li>
	</ol>
	</li>
	<li>Staining of hypotonic isolated vasculature
	<ol>
		<li>Block the hypotonic isolated vasculature 1 h with shaking at 100 rpm and room temperature with 500 &#181;L/well of 10 % donkey serum diluted in PBS.</li>
		<li>Incubate overnight at room temperature and shake at 100 rpm with 600 &#181;L/well of 1:100 PDGFR&#946; and 1:500 NG2 primary antibodies diluted in 10% donkey serum in PBS.</li>
		<li>Rinse the retinal network 3x in PBS for 5 min and incubate in 1:100 anti-mouse Alexa Fluor 594-linked and 1:100 anti-rabbit FITC-linked secondary antibodies diluted in 10% donkey serum in PBS shaking at 100 rpm and room temperature for 1 h in the dark.</li>
		<li>Rinse in PBS-T for 5 min and incubate in 0.2 ng/mL DAPI in PBS-T for 15 min followed by 3x 5 min rinses in PBS-T in the dark.</li>
		<li>Cut the tip of a plastic Pasteur pipet, moisten it with PBS-T and use it to transfer the retinal network to a 4-well glass chamber slide. 
		NOTE: The moistening step is important to avoid the retinal vasculature sticking to the inside of the Pasteur pipet.</li>
		<li>Unfold the retinal vasculature. Avoid touching the retinal vasculature with forceps as this can cause the vasculature to tangle. 
		NOTE: Unfolding can be done by tilting the chamber slide back and forth or aspirating drops of liquid onto the retinal vasculature.</li>
		<li>Remove the medium from the wells. The surface tension of the liquid will flatten the vasculature onto the bottom of the slide.</li>
		<li>Ensure correct unfolding under a microscope before removing the plastic wells from the chamber slide.</li>
		<li>Mount the stained vasculature with anti-fading mounting medium and a coverslip.</li>
	</ol>
	</li>
</ol>

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<li>Wittig, D., Jaszai, J., Corbeil, D., Funk, R. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+localization+and+characterization+of+putative+mesenchymal+stem+cell+markers+in+the+retinal+capillary+network+of+rodents%5BTitle%5D)+AND+%22Cells+Tissues+Organs%22%5BJournal%5D)">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>
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</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Geletin from porcine skin</td><td>Sigma-Aldrich</td><td>G2625-500G</td><td></td></tr><tr><td>Albumin from chicken egg white</td><td>Sigma-Aldrich</td><td>A5253-500G</td><td></td></tr><tr><td>Deoxyribonuclease (DNAse) I from bovine pancreas</td><td>Sigma-Aldrich</td><td>D5025-15KU</td><td>Dissolved in 0.15 M NaCl</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>VWR</td><td>0332-100G</td><td></td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch</td><td>017-000-121, lot 129348</td><td></td></tr><tr><td>Rabbit anti-PDGFR&amp;beta;</td><td>Santa Cruz</td><td>sc-432</td><td>1:100</td></tr><tr><td>Mouse anti-NG2</td><td>Abcam</td><td>ab50009</td><td>1:500</td></tr><tr><td>Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-585-152</td><td>1:100</td></tr><tr><td>Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>715-095-151</td><td>1:100</td></tr><tr><td>Cy2 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>711-225-152</td><td>1:100</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>715-165-150</td><td>1:100</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542-1MG</td><td>Dissolved in DMSO</td></tr><tr><td>Anti-fading mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Anti-fading mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Nunc Lab-Tek II 4-well chamber slide</td><td>Thermo Fisher Scientific</td><td>154526</td><td></td></tr></tbody></table>

retinal cryo-sections, whole-mounts, and hypotonic isolated vasculature preparations for immunohistochemical visualization of microvascular pericytes

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 PDGFRβ 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 capilla...

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 ...

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

Research

JoVE Journal

Biology

This article describes a method for labeling embryonic skin and thymus blood vessels.

A Method for Labeling Vasculature in Embryonic Mice

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

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

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

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.

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

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

Obtaining High-Quality Cryosections of Whole Rabbit Eye

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor β after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Cryosectioning and Immunostaining of Mouse Retina

The retina exhibits complex spatial organization across its laminar structure. Modern spatial transcriptomics and sequencing techniques require tissue sections &lt; 20 µm thick, limiting their retinal research applicability. This fresh-frozen cryosectioning method produces thin en face sections of mouse retina while preserving planar spatial relationships, enabling comprehensive molecular mapping across laminae.

En face Cryosectioning of Mouse Retina for High-dimensional Spatial Molecular Analysis

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.

Thin Sectioning of Slice Preparations for Immunohistochemistry

Learn three retinal tissue preparation techniques for immunohistochemical visualization of rat microvascular pericytes. Cryo-sections, whole-mounts, and hypotonic isolation enable detailed study of pericyte dynamics in ophthalmology research.

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes (Video) | JoVE Journal

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

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