3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Summary

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.

Abstract

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 β 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.

Introduction

Retinal pericytes are embedded within the basement membrane along the abluminal side of vascular endothelial cells, displaying a mesh-like pattern of short-range processes wrapped around the retinal blood vessels¹. This intimate contact between pericytes and the retinal vasculature contributes to maintaining retina environmental homeostasis². Although numerous studies have provided morphological evidence of pericytes in retinal vasculature, most of our understanding of the morphological characteristics of retinal pericytes comes from conventional histological techniques³.

Protocol

For this study, three young adult male C57BL/6 mice (8-10 weeks old, weighing 20-25 g) were used. They were housed in a 12 h light/dark cycle with controlled temperature and humidity and allowed free access to food and water. This study was approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. 2023-03-14-04) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academ.......

Discussion

In this study, we detailed the technical process for demonstrating the morphology of pericytes in retinal vasculature using retro-orbital injection of fluorescent tomato lectin, immunofluorescent examination with PDGFR-β, and solvent-based tissue clearing. The results show that these techniques are highly compatible for highlighting pericytes in the whole-mount retina. The combination of these methodologies offers an effective approach for visualizing the morphological characteristics of pericytes in retinal vascula.......

Materials

Name	Company	Catalog Number	Comments
4',6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	D3571	Protect from light
Alexa Fluor 488 donkey anti-goat IgG (H+L)	Thermo Fisher Scientific	A-11055	Protect from light
C57BL/6 mouse	Beijing Vital River Laboratory Animal Technology Co., Ltd.	SCXK (Jing) 2021-0006
Confocal imaging system	Olympus	FV1200
Goat anti-PDGFR-β	Research and Development	AF1042	25 µg
Imaris software	Oxford Instruments	v.9.0.1
Lycopersicon esculentum(Tomato) lectin, DyLight594	Thermo Fisher Scientific	L32471	1 mg
Microcentrifuge tube	Axygen	MCT-150-C	1.5 mL
Normal donkey serum	Jackson ImmunoResearch	017-000-121	10 mL
Panoramic tissue slice scanner	Olympus	VS120-S6-W
Photoshop and  Illustration	Adobe	CS6
RapiClear 1.52 Solution	SunJin Lab	RC152001	10 mL
Six-well plate	Costar	3335
Spring scissors	Fine Science Tools	15003-08
Superfrost Plus microscope slide	Thermo Fisher Scientific	4951PLUS-001	25 mm x 75 mm x 1 mm