Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Summary

The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

Abstract

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

Introduction

Blood vessels play an essential part of the vertebrate body as they provide the necessary nutrients, oxygen and hormonal signals to all organs. Also, since the discovery of their involvement in cancer development¹, they have received much attention in clinical research. Although a number of publications have investigated the mechanisms allowing blood vessel growth and morphogenesis, and a large number of genes important for their formation have been identified², a lot remains to be understood regarding the interaction between cells or tissues and the circulating blood.

Visualization of blood v....

Protocol

All animal handling was performed according to the recommendations for the care and welfare of research animals at the Norwegian University of Life Sciences, and under the supervision of authorized investigators.

1. Preparation of Instruments and Solutions

1. Prepare DiI stock solution dissolving 5 mg of DiI crystal in 1.5 mL plastic tube with 1 mL of 96 % EtOH. Vortex for 30 s and keep covered using aluminum foil.
   NOTE: The DiI stock solution can be conserved .......

Discussion

Cardiac perfusion with DiI previously has been used to label blood vessels in several model species²⁴, including teleost fish¹³_.

As DiI is directly delivered to the endothelial cell membrane by perfusion in the vasculature, it is possible to increase the signal-to-noise ratio by increasing the DiI concentration in the fixative solution. In addition, the fluorophore provides intense staining when excited with minimal bleaching allowing .......

Materials

Name	Company	Catalog Number	Comments
16% paraformaldehyde	Electron Microscopy Sciences	RT 15711
5 mL Syringe PP/PE without needle	Sigma	Z116866-100EA	syringes
BD Precisionglide syringe needles	Sigma	Z118044-100EA	needles 18G (1.20*40)
borosilicate glass 10cm OD1.2mm	sutter instrument	BF120-94-10	glass pipette
DiI (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)	Invitrogen	D-282
LDPE tube O.D 1.7mm and I.D 1.1mm	Portex	800/110/340/100	canula
Phosphate Buffer Saline (PBS) solution	Sigma	D8537-6X500ML
pipette puller	Narishige	PC-10
plastic petri dishes	VWR	391-0442
Super glue gel	loctite	c4356
tricaine (ms-222)	sigma	E10521-50G