Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Summary

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Abstract

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

Protocol

1. Collecting Mouse Embryonic Limb Skin (E13.5~E17.5)

1. Euthanize plugged females by approved procedure. According to our approved animal protocol, the females are euthanized by CO₂ exposure and then assured by cervical dislocation. Lay the animal on its absorbent paper towel and soak it thoroughly in 70% EtOH/H₂O from a squeeze bottle.
2. Dissect the uterus intact and place it in a 100 x 15 mm Petri dish containing ice-cold Hanks' Balanced Salt Solution (HBSS) to wash out blood.
3. Separate and dissect the embryo. Remove the very thin amnion from the embryo.
4. (Option) Dissect a single embryo in a 35 x 10 mm Petr....

Discussion

The vascular system is crucial for organ development during embryogenesis as well as for organ maintenance and reproductive functions in the adults, because it supplies sufficient oxygen and nutrients to the organs. Proper vascular network is well-established with complex and multi-step processes by angiogenesis in which pre-existing capillary network is reorganized with highly branched and hierarchical structures. Although numerous works have been shown that a variety of molecules is involved in these processes, it has .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies Pan-endothelial cell marker Antibody Species Company Catalogue # Working condition PECAM-1 Armenian hamster (M) Chemicon MAB1398Z 1:100 dilution#1 PECAM-1 Rat (M) BD 553369 1:300 dilution VEGFR2 Rat (M) eBioscience 14-5821-82 1:200 dilution CD34 Rat (M) eBioscience 13-0341 1:300 dilution Collagen IV Rabbit (P) AbD Serotec 2150-1470 1:300 dilution#2 Arterial endothelial cell marker Antibody Species Company Catalogue # Working condition Neuropilin1 Rabbit (P) The Alex Kolodkin’s lab#3 1:3000 dilution Unc5H2 Goat (P) R&D AF1006 1:200 dilution Venous endothelial cell marker Antibody Species Company Catalogue # Working condition EphB4 Goat (P) R&D AF446 1:100 dilution Lymphatic endothelial cell marker Antibody Species Company Catalogue # Working condition LYVE-1#4 Rabbit (P) Abcam ab14917 1:200 dilution LYVE-1#4 Rat (M) MBL D225-3 1:200 dilution Prox-1 Rabbit (P) Chemicon AB5475 1:1000 dilution Prox-1 Goat (P) R&D AF2727 1:50 dilution Neuropilin2 Rabbit (P) Cell Signaling 3366 1;100 dilution Podoplanin Syrian hamster (M) Hybridoma Bank 8.1.1 1:200 dilution Smooth muscle cell/pericyte marker Antibody Species Company Catalogue # Working condition αSMA-Cy3 Mouse (M)#5 Sigma c-6198 1:500 dilution#6 NG2 Rabbit (P) Chemicon AB5320 1:200 dilution SM22α Rabbit (P) Abcam ab14106 1:200 dilution Antibodies forGFP reporter Antibody Species Company Catalogue # Working condition GFP Rabbit (P) Invitrogen A11122 1:300 dilution GFP Rat (M) Nacalai tesque 04404-84 1:1000 dilution GFP Chick (P) Chemicon P42212 1:300 dilution Antibodies for LacZ reporter Antibody Species Company Catalogue # Working condition β-gal Rabbit (P) MP Biomedical 55976 1:5000 dilution β-gal Goat (P) AbD Serotec 4600-1409 1:500 dilution β-gal Chick (P) Abcam ab9361 1:200 dilution Antibodies for peripheral axon Antibody Species Company Catalogue # Working condition 2H3 Mouse (M)#7 Hybridoma Bank 2H3 1:200 dilution Tuj1 Mouse (M)#8 Covance MMS-435P 1:500 dilution Peripherin Rabbit (P) Chemicon AB1530 1:1000 dilution Antibodies for migrating Schwann cells BFABP Rabbit (P) The Thomas Müller’s lab#9 1:3000 dilution (P): polyclonal antibody, (M): monoclonal antibody #1: Goat anti-Armenian hamster-Cy3 (Jackson ImmunoResearch 127-165-160) antibody should be used as a secondary antibody. #2: The Collagen IV antibody can be used to detect blood vessels after in situ hybridization. #3: The Neuropilin1 antibody is kindly provided by the Alex Kolodkin’s lab in the Johns Hopkins University. Sheep anti-human Neuropilin1 antibody is available in R&D (AF3870), although we have not tested it yet. #4: The LYVE-1 antibodies also detect a subset of macrophages in the embryonic skin. #5: The anti-αSMA antibody is mouse IgG2a monoclonal antibody. #6: The Cy3-conjugated αSMA antibody is incubated for 1 hour at room temperature together with secondary antibodies for other primary antibodies. #7: 2H3 antibody is mouse IgG1 monoclonal antibody against neurofilament. #8: Tuj1 antibody is mouse IgG2a monoclonal antibody against Neuron-specific class III beta-tubulin. #9: The BFABP (brain-specific fatty acid binding protein) antibody is kindly provided by the Thomas Müller’s lab in Max-Delbrück-Center for Molecular Medicine.