Name: retinal cryo-sections, whole-mounts, and hypotonic isolated vasculature preparations for immunohistochemical visualization of microvascular pericytes
Uploaded: 2018-10-07T14:00:00.000+00:00
Duration: 10 min 46 s
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>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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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 unnecessary animal sacrifice. The preparatory work is decisive for good results. It is crucial while dissecting the eye not to pull the eye but to cut all the muscles around the eye. There is a risk of retinal detachment if force is used. Because the eye cup is thin, one should not fix too long. Immersion with 4% paraformaldehyde in PBS for 2&#8211;4 h will fix the tissue. If fixed longer, there is a risk of destroying the antigen of interest. In order to generate the best possible immunostainings, it is critical for each section to be as flat as possible against the glass. Because the sections contain only a few occasional transverse cuts of the microvasculature, this tissue preparations technique offers only intermittent visualization of the microvasculature. Therefore, this method is not suitable for the studies of the overall vascular architecture and quantifiable measures of pericytes.
The overall vasculature can be stained after whole-mounting to provide an overview of the vasculature. When mastering this technique, it can be a fast tissue preparation procedure that requires relatively little workload. However, there are a few points to be particularly aware of. The retina is fragile and it is paramount to handle them with care and avoid rips and cuts. Furthermore, while carefully handling the retina, it is of key importance to avoid folds and other crevasses during mounting that may affect the immunohistochemical imaging later on. The mounted retina has a thickness of 250-300 &#181;m and antibody penetration of the neuronal layers might be problematic. In regard to pericyte visualization, the NG2 staining in the whole-mount protocol could only be achieved with a Cy3-linked secondary antibody, and PDGFR&#946; immunoreactivity in the whole-mounted adult retina resulted in such a very weak signal that it was not shown in the figure. Hence, the usefulness of the whole-mount staining technique is strongly hampered by the successful staining depending mainly on the specific primary and secondary antibodies used. Further suggestions to optimize the protocol for visualized microvascular plexuses of an adult rat retina is by the incubation in primary antibody for a longer period of time and/or changing the incubation temperature26.
Due to the thickness of the whole-mounted, it is important to be aware of the direction of the retina after making the four incisions from the retinal periphery towards the optic nerve head to avoid accidental mounting with the inner retina containing the capillaries facing down. Still, detailed visualization of the deeper microvascular plexuses and the pericytes can be troublesome with this method14,20. As whole-mount staining of the adult retina is difficult in the deeper capillary plexus, this might result in the retina appearing avascular in some parts. The immunohistochemical staining of whole-mounted adult retina provides only a fragmented overview of the vasculature with pericytes lining the vessels in the superficial capillary plexus and therefore, this method can lead to false negative results in the visualization of the rat retinal vasculature.
Trypsin digestion has long been considered the gold standard technique for isolation and visualization of the retinal vasculature20. Hypotonic isolation provides an alternative method for the visualization of the entire complex three-dimensional vascular network and faces many of the same challenges working with very fragile tissue which is difficult to handle. Due to the resemblance in the retinal vascular product between the two methods, the hypotonic isolation faces many of the same challenges as trypsin digestion20 including careful dissection to prevent major tears and cautious handling by avoiding pipetting and touching with forceps. An important difference between the methods is the fixation. Trypsin digestion is typically done after fixation and can be applied successfully on preserved retinas that have been in fixative for several years20. Hypotonic isolation is performed before fixation and thus allows for the implementation in various other assays as previously described in literature23,24,25. The trypsin digestion is performed over two days20, whereas the hypotonic isolation can be completed within hours. Furthermore, the hypotonic isolation protocol does not include the risk of over-digestion and enzymatic cleavage of membrane markers required for pericyte recognition, e.g. PDGFR&#946; and NG2, making this method preferred for subsequent immunohistochemical microvascular staining.
Finally, although out of the immediate scope of this manuscript, there is a less widely known but very useful technique for isolating large, intact microvascular plexi from rodent out of the retina27. The retina is positioned in a special glass chamber and coverslip. Upon the removal of the coverslip, the microvasculature remains attached to the coverslip to create a fully alive (&#8776; 98% of cells) tissue print. The biggest advantage of this method is that it allows for the physiological study of the vessels and subsequent fixation for immunostaining. An obvious limitation of the method is that it does not provide the visualization/isolation of the entire microvascular network as retina flat mount and hypotonic preparations do. Still, pericytes can be visualized by application of this method27.
The three tissue preparation techniques described here are complementary in the sense that each method comprises advantages and shortcomings for visualization of the microvascular pericytes. The assessment of each methods potentials and weaknesses is important to select the optimal method for investigating the microvascular pericytes under specific pathological conditions. The demonstrated methods can be expanded to staining not only for pericyte markers but also for the visualization of microvascular structures in general. Eventually, it is for the individual researcher to select the appropriate visualization method based on the research hypothesis in question.

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 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 capillary plexuses contain pericytes that display PDGFR&#946; and NG2 immunoreactivity (Figure 1). The unique position of pericytes in the vascular wall can be seen as circular or horseshoe-shaped immunoreactivity in the periphery of the vessels (Figure 1, arrows) or occasional longitudinally cut vessels (Figure 1, arrowhead).
Successful visualization of the microvascular pericytes in whole-mount preparation of adult rat can be challenging14,20. PDGFR&#946; immunoreactivity in the whole-mounted adult retina resulted only in a very weak signal (not shown). NG2 staining outlined the inner vascular network with intense staining in abluminal pericytes (Figure 2).
To obtain an overview of the vascular network of the adult rat retina, immunohistochemistry of hypotonic isolated rat retinal vascular network provides an alternative method to whole-mount. When successfully performed, this tissue preparation technique provides an overview of the entire retinal microvasculature including inner and deep capillary plexuses. Immunohistochemical staining with pericytic markers results in the entire microvascular network showing NG2 immunoreactivity with an intense response in specific vascular cells (Figure 3). Some of these cells also display PDGFR&#946; immunoreactivity (Figure 3).
A careful unfolding of the tissue is important to obtain a good overview of the retinal vasculature. Insufficient unfolding can result in the vasculature being arranged in multiple focus layers which makes fluorescent imaging difficult (Figure 4). Unfolding can be facilitated by gentle shaking, tilting the chamber slide back and forth or aspirating drops of liquid onto the retinal vasculature. It should be noted that the anatomy of the retinal capillary plexuses will result in some overlay of the vascular beds. Hence, it is problematic to distinguish the inner from the deeper capillary plexuses when flattened onto the glass slide.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57733/57733fig1.jpg" /> 
Figure 1: Double immunohistochemical pericyte staining of cryo-section of the inner rat retina. (A)&#160;NG2 (green) 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. (B) PDGFR&#946; (red) immunohistochemistry show reactivity similar to NG2 immunoreactivity. Arrows and arrowhead point at the same structures as for NG2 staining. (C) The image shows a merge of NG2, PDGFR&#946; and DAPI (blue) by superimposing the three different filters. It is revealed that the two antibodies are co-localized (green + red = yellow). NF: nerve fiber layer, GCL:&#160;ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL : outer plexiform layer, ONL: outer nuclear layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57733/57733fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57733/57733fig2.jpg" /> 
Figure 2: Immunohistochemical pericyte staining of whole-mounted rat retina. NG2 (red) immunoreactivity is visible along the superficial vascular networkwith intense staining in abluminal pericytes. The images show merged NG2 and DAPI (blue). The neuronal cells are visible as DAPI-blue nuclei between the NG2-stained microvasculature. PDGFR&#946; immunoreactivity in the whole-mounted adult retina resulted only in a very weak signal and is therefore not included in this figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57733/57733fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57733/57733fig3.jpg" /> 
Figure 3: Double immunohistochemical pericyte staining of hypotonic isolated rat retinal vasculature. (A). The image shows hypotonic isolated rat retinal vascular network immunohistochemical stained with NG2 (green) and PDGFR&#946; (red). The complete network showed NG2 immunoreactivity. However, PDGFR&#946; immunoreactivity was found in cell somas. The insert of A is an overview of retinal vasculature network stained with DAPI (blue). (B) The image shows the same specimen as (A) at a different location and higher magnification. It is clear that PDGFR&#946; staining is only displayed in cell somas in the vascular network indicating pericyte immunoreactivity. The insert of B is the image that shows high magnification of vasculature stained with NG2 (green), PDGFR&#946; (red) and merged with DAPI. Including DAPI staining together with NG2 and PDGFR&#946; reveals three pericytes (PDGFR&#946;-positive) and two unidentified vessel wall cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57733/57733fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57733/57733fig4.jpg" /> 
Figure 4: Example of an inadequate unfolded hypotonic isolated retinal vasculature. The careful unfolding of a hypotonic isolated vasculature is important as insufficient unfolding can result in the vasculature being stacked. The pictures show DAPI-stained retinal vasculature at the same site but taken in different focus layers. (A)&#160;The DAPI-stained cells in one focus layer show an approximately 25 &#181;m thick blood vessel in the retinal vasculature. (B)&#160;The DAPI-stained cells in a second focus layer elucidate thin branching capillaries at the same site that has been stacked on the larger blood vessel displayed in (A). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57733/57733fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

10.0K Views.  Glostrup Research Institute. Deze methoden kunnen helpen bij het beantwoorden van belangrijke vragen op het gebied van oogheelkunde, zoals pericytenproliferatie of migratie. Het belangrijkste voordeel van deze procedure is dat ze een verscheidenheid aan visualisatieopties bieden. Het aantonen van de procedures zijn Karin Dreisig en Frank W.Blixt, twee post-docs in ons laboratorium.Om het oog teucleren, gebruik je eerst een scalpel om de achterste en voorste spleten van ongeveer 0,5 centimeter in het rattenooglid t...

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

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

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

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Immunology and Infection

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Neuroscience

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

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

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

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.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioengineering

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; 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.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

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.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

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

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

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

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

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

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

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