Target Discovery and Validation Reviews and Protocols

In Vivo Assay of Human Angiogenesis 263
and 0.25 µg/mL amphotericin B) at 37°C in a humid 95% air, 5% CO 2 atmosphere
and split the cells at ratios 1:3.
3.2. Animal Experiments
3.2.1. Transfer of Human Endothelial Cells Into RAG2 Knockout Mice
1. Trypsinize subconfluent HUVECs, wash, and count. Pellet the cells (3 × 10 5 cells
per plug) by means of another centrifugation and discard all extra medium from
the tube (see Note 2).
2. Thaw Matrigel on ice and adjust it to a final protein concentration of 8 mg/mL
with sterile PBS. Add 200 µL of Matrigel per plug to the cell pellet and mix gently
to avoid foaming.
3. Anesthetize RAG2 knockout mice of 4–12 wk of age by injection of 150 µL of a
saturated mixture of hypnorm + dormicum in the abdominal cavity.
4. Inject the Matrigel/HUVEC mix (200 µL per plug) subcutaneously on the abdominal
midline of the anesthetized animal with a 23-gauge needle (Fig. 2A,B; see Note 3).
Leave the mouse on its back to allow gelation of the Matrigel as it will rapidly gel
at room temperature (Fig. 2C). It is also possible to inject up to four gels into one
mouse: one on either side of the abdominal midline and one laterally to the former
in either flanks.
5. Sacrifice the mice by cervical dislocation upon termination of the experiment.
3.2.2. Implantation of Endostatin Sontaining Microosmotic Pumps
1. Fill Alzet microosmotic pump (model 1002 for experiments up to 14 d or model
2004 for up to 28 d) with a 7.9 mg/mL solution of recombinant endostatin.
2. Anesthetize mice with Hypnorm/Dormicum.
3. Wash the neck with 70% ethanol.
4. Make a small midline incision in the skin and carefully make a subcutaneous
pocket by blunt dissection towards the tail, large enough to accommodate the
pump.
5. Insert the pump and make sure the edges of the skin adapt without strain. If necessary,
extend the pocket to relieve pressure from the implant (Fig. 2D,E).
6. Close the wound with nonabsorbable suture (Fig. 2F).
7. Carefully inspect the wound 1 d postinjection for signs of inflammation and infection
before proceeding to injection of the Matrigel plugs.
3.3. Histology and Immunohistochemical Analysis of Matrigel Plugs
3.3.1. Methods for Processing and Staining
1. Sacrifice the mice and use scissors to gently cut out the abdominal wall around the
injected Matrigel plug, making sure to include all tissue layers. Transfer the harvested
sample to PBS on ice. Fix samples in methanol or 10% formalin (4°C; 24 h),
or snap-freeze them in OCT in liquid nitrogen.
2. Embed the methanol- or formalin-fixed tissues into paraffin and cut 4-µm-thick
sections. Deparaffinize and rehydrate the methanol-fixed sections in successive