Target Discovery and Validation Reviews and Protocols

260 Skovseth, Küchler, and Haraldsen
10. Nikon E-800 fluorescence microscope (Nikon).
11. F-view digital camera controlled by the AnalySIS 3.2 Software (Soft Imaging
Systems GmbH).
12. Reagents for H&E staining (hematoxylin, hexamine, azophloxine, saffron, and
ethanol).
13. Phosphate-buffered saline (PBS).
14. Bovine serum albumin to dilute antibodies (1.25% for primary reagents and 12.5%
for secondary reagents).
15. SuperFrost or chromalun-coated glass slides.
16. 10% Formalin.
2.3.2. Primary Antibodies (Clone, Working Dilution,
Specification, Source)
1. hCD31 (hec7, 0.4 µg/mL, mouse IgG2a, W. A. Muller, New York).
2. hICAM-1 (RRI/1.1.1, 10 µg/mL, mouse IgG1, R. Rothlein, Ridgefield, CT).
3. hVCAM-1 (hec7, 0.4 µg/mL, mouse IgG2a, W. A. Muller, New York).
4. hE-selectin (hec7, 0.4 µg/mL, mouse IgG2a, W. A. Muller, New York).
5. mCD31 (Mec 13.3, 1/100, rat IgG2a, A. Vecchi, Milan).
6. α-Smooth muscle actin, α-SMA (M0851, 10 µg/ml, mouse IgG2a, DakoCytomation
www.dako.com).
2.3.3. Secondary Reagents
1. Alexa 594-conjugated goat anti-rat IgG (10 µg/mL, Invitrogen).
2. Alexa 546-conjugated goat anti-mouse IgG (1 µg/mL, Invitrogen).
3. Fluorescein isothiocyanate (FITC)-conjugated Ulex europaeus lectin (1/400,
Vector Laboratories).
4. Rhodamine-conjugated phalloidin (1/200, Sigma).
2.4. Re-isolation of Endothelial Cells, mRNA Purification, and Analysis
1. Collagenase (cat. no. C8167, Sigma).
2. Dispase II (Roche Diagnostics).
3. DNaseI (Roche Diagnostics).
4. 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, and 0.25 µg/mL
amphotericin B).
5. 40-µm Filter (Falcon).
6. Rock’n roller.
7. Antibody to hCD31 (hec7).
8. Dynabeads M-450 coated with sheep anti-mouse IgG (Dynal Biotech).
9. Dynal MPC-6 magnet (Dynal Biotech, Oslo).
10. RNeasy mini kit (QIAGEN GmbH).
11. Dithiothreitol (DDT), oligo(dT), and Superscript III reverse transcriptase
(Invitrogen).
12. Taq polymerase and buffer, dNTPs.