Authors: Gargioli Cesare 

### Introduction 

With this technique it is possible to label the blood vessel in vivo in order to analyze in whole mount the quantity and quality of blood vessel.

### Reagents 

1. 1.0 ml syringe
- China Black Ink
- Stadard anesthetic (Ketamine and Xylazine)

### Procedure

1. Anesthetize the mouse
- Using a 1 ml syringe withdraw 0,5 ml of mouse blood from the heart ventricle
- Inject 1 ml of China Ink and wait for 30 sec (until the eyes, mouth and legs became black)
- Quickly dissect and fix in 4% PFA over night at 4°C
- Treat the sample with a solution of 0,5% KOH for one day
- Treat the sample with a solution of 0,5% KOH/Glycarol 2:1 one day
- Treat the sample with a solution of 0,5% KOH/Glycerol 1:1 for one day
- Treat the sample with 100% Glycerol for one day (in order to visualize clearly all the inner blood vessels)

### Timing 

4 days

### Critical Steps
 
Injection in the heart after blood withdrawal

### Anticipated Results
 
eyes, mouth and legs black stained 

### Figures
 
![Fig 1](http://i.imgur.com/HLXmpD2.png "Fig 1")

### Associated Publications 

**PlGF–MMP-9–expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle**, Cesare Gargioli, Marcello Coletta, Fabrizio De Grandis, Stefano M Cannata, and Giulio Cossu, *Nature Medicine* 14 (9) 973 - 978 27/07/2008 [doi:10.1038/nm.1852](http://dx.doi.org/10.1038/nm.1852)

### Author information
 
**Gargioli Cesare**, S. Raffaele Biomedical Park Foundation 

Correspondence to: Gargioli Cesare (cegargioli@yahoo.it) 


*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/467) (2008) doi:10.1038/nprot.2008.161. Originally published online 30 July 2008*.