Target Discovery and Validation Reviews and Protocols

264 Skovseth, Küchler, and Haraldsen
baths of xylene (5 min; 20°C), 100% ethanol (1 min; 20°C), 96% ethanol (1 min;
20°C), 70% ethanol (1 min; 20°C), and finally PBS (2 min twice; 20°C) before
staining.
3. Cut frozen sections on a cryotome (4 µm) and fix in acetone (20°C; 15 min) before
staining.
4. Stain methanol-fixed and the acetone-fixed sections with H&E for histological
analysis to facilitate identification of Marigel plug.
5. Stain the frozen sections with primary and secondary antibodies with each 1-h
incubation step at room temperature for immunohistochemistry. Wash between
these steps in PBS for 1 min.
6. Stain the deparaffinized methanol- or formalin-fixed sections with the primary antibody
(overnight), followed by a 3–h incubation with a secondary fluorochromeconjugated
reagent for immunohistochemistry. Wash between these steps in PBS
for 10 min.
7. For two-parameter analysis, add rhodamine-conjugated phalloidin or FITCconjugated
Ulex lectin to the primary antibody solution.
3.4. Harvesting of Human ECs, mRNA Isolation, and Expression
Analysis of Human Endothelium
3.4.1. Isolation of Human Endothelium From Matrigel Plugs
1. Dissect Matrigel plugs from the skin and muscle layers (Fig. 2G, H) and cut into
small pieces with a scalpel on ice.
2. Incubate the Matrigel fragments in a mixture of 0.1% collagenase, 0.2% dispase,
and 20 µg/mL DNaseI in PBS at 37°C for 40 min on a magnet stirrer (see Note 4).
3. Wash the cell suspension in 40 mL of cold complete MCDB-131.
4. Filter the cell suspension through a 40-µm sieve filter to remove debris.
5. Incubate 1 mL of cell suspension with a primary mouse antibody to 0.3 µg/mL
hCD31 (15 min on ice).
6. Wash the cell suspension in 15 mL of cold complete MCDB-131.
7. Incubate 1 mL of the cell suspension with sheep anti-mouse immunoglobulincoated
Dynabeads according to the recommendation of the manufacturer.
8. Dilute the cell suspension to 5 mL and extract the rosetting cells on a Dynal magnet.
Wash the cells by repeating the extraction of rosetting cells five times.
9. Spin down the cells and isolate RNA.
3.4.2. Isolation of RNA
1. Isolate total RNA and perform on-column DNaseI treatment by using the RNeasy
mini kit according to instructions of the manufacturer.
2. Reverse transcribe 200 ng of total RNA by using oligo(dT) and the Superscript III
reverse transcriptase kit according to the manufacturer’s instructions. In brief,
mix RNA, dNTPs, buffer, DDT, oligo(dT), RNasin, and enzyme to a final
volume of 20 µL and incubate for 1 h at 50°C and subsequently for 15 min
at 70°C.