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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 cryo....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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