A Method for Labeling Vasculature in Embryonic Mice

Summary

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

Abstract

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-β 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.

Protocol

1. Fluorescein labeled dextran and GSL I-isolectin B₄ facial vein injections to label embryonic vasculature

1. Prepare FITC-dextran (50ug/mL) in Phosphate Buffered Saline (PBS) or GSL 1 - isolectin B₄ (20ug/200uL) in PBS in a 1.5mL Eppendorf tube and warm to 37°C. Add 100uL of stock 1.25mM Fast Green/PBS to the FITC-dextran solution (total volume 1mL) and 180uL of stock 1.25mM Fast Green/PBS to the GSL 1 - isolectin B₄ (total volume 200uL), so that the solution is visibly blue.
2. Dissect E14.5-E18.5 embryos and yolk sac together, leaving the allantoic stalk (umbilical artery and vein) intact.
3. Transfer embryos....

Discussion

Whole-mount and PECAM-1 (CD31) staining on sections are the conventional methods for labeling the vasculature in embryonic mice. These methods require the use of direct and/or indirect immunofluorescence, and detergents to permeabilize mouse tissue. This proves to be a rather timely process. Here, we have employed FITC-dextran or isolectin B₄ facial vein injections to directly label the embryonic vasculature, thereby eliminating the requirement for antibody labeling steps. Furthermore, this method allows the a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FITC-dextran	Sigma-Aldrich	FD150S-1G
Fluorescein labeled GSL 1 - isolectin B4	Vector Laboratories	FL-1201
Fast Green	MP Biomedicals	195178
PFA	Fluka	76240
Fetal Bovine Serum	Atlanta Biologicals	S11550
Optimal Cutting Temperature Compound (O.C.T.	VWR international	25608-930
Acetone	JT Baker	9006-33
Donkey Serum	Jackson	017-000-121
rat anti-mouse CD31,	BD Biosciences	558736
goat anti-mouse PDGFR-β	R&D Systems	AF1042
donkey anti-rat CD31 Alexa 647 (Invitrogen)	Biolegend	102516
donkey anti-goat Alexa 594 (Invitrogen)	Invitrogen	A11058
Triton X -100	Sigma-Aldrich	X-100
Low melt agarose/PBS	Sigma-Aldrich	A9414-25G
Methanol	Fisher Scientific	A413-4
Benzyl Alcohol	Acros Organics	148390010
Benzyl Benzoate	Acros Organics	105860010
Depression slides	Fisher Scientific	S175201
Fluorogel	Electron Microscopy Sciences	17985-10
Cover Glass (22X22)-1.5	Thermo Fisher Scientific, Inc.	152222
Zeiss LSM 510 Meta Confocal Microscope	Carl Zeiss, Inc.
Micro dissecting forceps	Roboz Surgical Instruments Co.	RS-5135
Parafilm No. OM992	Fisher Scientific	13-374-16
12 and 24 well microplates	Evergreen Scientific	222-8044-01F
Superfrost/PlusMicroscope Slides	Fisher Scientific	12-550-15
4mL clear vials	National Scientific Company	B7800-2