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

Podsumowanie

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.

Streszczenie

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 β (PDGFRβ) 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.

Wprowadzenie

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 layers^4,5. 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 β (PDGFRβ) 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 PDGFRβ/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 ischemia^10,11). The vasculature is evident as transverse cuts through the retina, making it possible to separately study the capillary plexuses within the respective retinal layers^12,13.

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 retina^{14,15,16,17,18,19}. 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 degeneration^20,21,22. 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 blotting^23,24,25. 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.

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Protokół

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

1. Cryo-section
   1. Make posterior and anterior ~0.5 cm slits of in the rat eyelid with a scalpel.
      1. 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.
   2. 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.
   3. 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.
   4. 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 2–4 h.
   5. Rinse sequentially in Sörensen's phosphate buffer with 10% sucrose and 25% sucrose.
   6. 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 °C.
2. Whole-mount
   1. Make posterior and anterior ~0.5 cm slits in the rat eyelid with a scalpel.
   2. 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.
   3. 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.
   4. Under a microscope, cut along the corneal limbus with dissection scissors to remove the cornea and remove the lens with forceps.
   5. Separate the retina from the retinal pigment epithelium towards the optic nerve with forceps using small opening movements to avoid major tearing.
   6. 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.
   7. Spread the retina onto a glass slide and allow drying for 5–10 min.
   8. Fix in 4% formaldehyde for 20–30 min by dripping formaldehyde onto the retina.
      CAUTION: Do not apply directly on the retina as it may detach from the glass.
   9. Rinse with PBS. For optimal results, immuno-stain directly after rinsing.
3. Hypotonic isolation
   1. Make posterior and anterior ~0.5 cm slits in the rat eyelid with a scalpel.
   2. 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.
   3. Make an initial hole at the corneal limbus by applying light pressure with the tip of a scalpel.
   4. Under a microscope, cut along the corneal limbus with dissection scissors to remove the cornea and remove the lens with forceps.
   5. Separate the retina from the retinal pigment epithelium towards the optic nerve head with forceps using small opening movements to avoid major tearing.
   6. 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.
   7. 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.
   8. Rinse minimum 3 times in deionized water for 5 min with shaking at 150–300 rpm to remove neuronal cell debris. The retina should become more transparent with each rinse indicative of the removal of neuronal cellular debris.
      1. Use a dark background to look into the 24-well plate to clearly see the diaphanous isolated retinal vasculature.
      2. (Optional): 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.
   9. 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 °C.

2. Immunohistochemistry

1. Staining of cryo-sections
   1. Cut 10 µm 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).
   2. Submerge the glass slide in PBS with 0.25 % Triton X-100 (PBS-T) for 15 min.
   3. Drip 1:100 PDGFRβ and 1:500 NG2 primary antibodies diluted in PBS-T + 1% BSA onto the cryo-section and incubate in incubation chambers at 4 °C overnight.
   4. 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.
   5. Incubate the glass slide 1 h at room temperature in the dark.
   6. 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.
   7. Mount the stained cryo-sections with anti-fading mounting medium containing DAPI and a coverslip.
2. Staining of whole-mount
   1. Drip PBS-T onto the whole-mount and incubate at room temperature for 15 min.
   2. Pour off and drip 1:100 PDGFRβ and 1:500 NG2 primary antibodies diluted in PBS-T + 1% BSA and incubate in moist chambers at 4 °C overnight.
   3. Pour off and drip on PBS-T to rinse the glass slide in 2x 15 min.
   4. 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.
   5. 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.
   6. Mount the stained whole-mount with anti-fading mounting medium containing DAPI and a coverslip.
3. Staining of hypotonic isolated vasculature
   1. Block the hypotonic isolated vasculature 1 h with shaking at 100 rpm and room temperature with 500 µL/well of 10 % donkey serum diluted in PBS.
   2. Incubate overnight at room temperature and shake at 100 rpm with 600 µL/well of 1:100 PDGFRβ and 1:500 NG2 primary antibodies diluted in 10% donkey serum in PBS.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. Remove the medium from the wells. The surface tension of the liquid will flatten the vasculature onto the bottom of the slide.
   8. Ensure correct unfolding under a microscope before removing the plastic wells from the chamber slide.
   9. Mount the stained vasculature with anti-fading mounting medium and a coverslip.

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Wyniki

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 ca...

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Dyskusje

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...

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Materiały

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