Target Discovery and Validation Reviews and Protocols

266 Skovseth, Küchler, and Haraldsen
2. Place slides in Ventana robot and activate the hybridization protocol. Slides will
be deparaffinized and pretreated for enhanced detection. The probes that are
described here were most efficiently visualized by choosing a mild protocol for
target retrieval (alkaline protease 3 and Ribo CC).
3. Add 100 µL of the sense or antisense dilutions to the sections. The hybridization
is performed at 60°C (6 h) with three stringency washes in 2X SSC (6 min; 60°C).
Riboprobes are detected with biotinylated 2.2 µg/mL anti-digoxin (32 min; 37°C)
and the BlueMap kit.
4. Retrieve the slides from the robot and wash the hybridized sections in hot water to
remove the oil.
3.5.3. Double Staining With Ulex Lectin
1. Wash freshly hybridized sections after removal of oil in baths of 70% alcohol
(1 min; 20°C), 96% alcohol (1 min; 20°C), 100% alcohol (1 min; 20°C), xylene
(2 min; 20°C) (see Note 5), 100% alcohol (1 min; 20°C), 96% alcohol (1 min;
20°C), 70% alcohol (1 min; 20°C), and finally PBS (2 min twice; 20°C).
2. Incubate the sections with FITC-labeled Ulex lectin for 1 h at room temperature.
3. Wash the sections in PBS (2 min twice) and mount in PVA (see Note 5).
4. Notes
1. Endostatin released from the osmotic pumps can be detected throughout the experimental
period as demonstrated by analysis of endostatin in serum by using an
ELISA kit from CytImmune Sciences (19).
2. In our studies, we have used freshly isolated confluent HUVECs in low passage
(passage 1–4 is preferable). A pool of three individual cell cultures in low passage
gives reproducible results in the model system.
3. At all time-points before injection, keep the Matrigel and the Matrigel/HUVEC
mix on ice. It is also important to gently mix the Matrigel/EC suspension in the
syringe before injection to avoid sedimentation of ECs. This mixing can be done
by letting a small air bubble travel through the gel in the syringe.
4. It is important to use sufficient amounts of the collagenase/dispase solution to
completely dissolve the Matrigel. Use at least 6 mL of solution for one to five
plugs and 10 mL of solution for six to 12 plugs.
5. Usually, in situ-hybridized sections would be mounted in Eukitt for greater stability
of the 5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) signal, but this is
not favorable for the fluorescent FITC signal. We therefore chose to mount the
stained sections with PVA, which is the standard mounting medium for fluorescent
signals. Because the PVA does not stabilize the BCIP signal, we recommend
analyzing the sections immediately.
References
1. Risau, W. (1997) Mechanisms of angiogenesis. Nature 386, 671–674.
2. Carmeliet, P. (2000) Mechanisms of angiogenesis and arteriogenesis. Nat. Med.
6, 389–395.