Target Discovery and Validation Reviews and Protocols

In Vivo Assay of Human Angiogenesis 261
13. PDGF-β LightCycler kit (Search-LC).
14. Human PDGFB-specific PCR primers 5′-CAT AGA CCG CAC CAA CGC CAA
CTT C-3′ as forward primer and 5′-ATC TTT CTC ACC TGG ACA GGT CGC
AG-3′ as reverse primer.
15. Human hydroxymethylbilane synthase-specific PCR primers 5′-GTA ACG GCA
ATG CGG CTG-3′ as forward primer and 5′-GGT ACG AGG CTT TCA ATG-3′
as reverse primer.
16. QuantiTect SYBR Green PCR kit (QIAGEN GmbH).
17. LightCycler (Roche Diagnostics).
2.5. In Situ Hybridization
2.5.1. Probe Generation and Labeling
1. A chloramphenicol-resistant pBC Vector containing a 1-kb insert of hPDGFB
was kindly provided by C. Betsholtz (Department of Medical Biochemistry and
Biophysics, Karolinska Institute, Stockholm, Sweden).
2. EcoRI and NotI restriction enzymes (New England Biolabs).
3. Phenol/chloroform extraction: phenol saturated TE8, phenol isoamyl alcohol,
sodium acetate, 96% ethanol, and 70% ethanol.
4. DIG RNA labeling kit (Roche Diagnostics).
5. T7 and T3 RNA polymerases (Roche Diagnotics).
6. Ribohybe (Ventana Medical Systems).
2.5.2. In Situ Hybridization
1. Ventana Discovery automated histology robot (Ventana Medical Systems).
2. Ventana solutions as supplied by Ventana Medical Systems: mild alkaline protease
3, Ribo CC, standard saline citrate (SSC), and BlueMap kit for developing the nitroblue
tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate alkaline phosphatase
substrate.
3. Biotinylated anti-digoxin antibody (Jackson ImmunoResearch Laboratories).
4. DIG-labeled RNA probe.
2.5.3. Double Staining of In Situ Hybridized Sections With Ulex Lectin
1. 70, 96, and 100% ethanol baths.
2. Xylene bath.
3. FITC-labeled Ulex lectin and polyvinyl alcohol (PVA).
3. Methods
3.1. Cell Preparation
1. Isolate HUVECs from the umbilical vein with 0.2% collagenase as described by
Jaffe et al. (32).
2. Culture HUVECs in complete MCDB-131 (containing 7.5% fetal calf serum,
10 ng/mL rhEGF, 1 ng/mL rhbFGF, 1 µg/mL hydrocortisone, 50 µg/mL gentamicin,