AMPK Methods and Protocols
- Cool all required solutions and materials to 4C (PBS, pro-
and antiangiogenic compounds, 15 mL tubes, 2 mL tubes,
pipette tips, 1 mL syringes, 20- and 27-gauge needles) (see
Note 45). All solutions and materials have to be sterile. Work
under a laminar flow hood.
- After thawing, mix the Matrigel carefully by slowly pipetting it
up and down ten times with precooled tips (seeNote 46).
Prepare 1.5 mL aliquots in 2 mL tubes.
- Add the required agents (growth factors, inhibitors) or vehicles
to the Matrigel solution; vortex and spin briefly in a precooled
4 C centrifuge (seeNote 47). All solutions have to be kept at
4 C before being added to Matrigel. VEGF is applied at a final
concentration of 400 ng/mL together with 400μg/mL of
heparin (seeNote 48).
- Fill 500μL of Matrigel with or without added compounds into
a 1 mL syringe using a 20-gauge needle. Keep the syringe with
Matrigel flat on ice until use (seeNote 49).
- Determine the body weight of each mouse, and inject a mixture
of anesthetics intraperitoneally (final concentrations: 0.5 mg/
kg medetomidine, 0.05 mg/kg fentanyl, 5 mg/kg
midazolam).
- Disinfect the flanks of the mice with 70% ethanol using a Q-tip.
- Remove the Matrigel-containing syringe from the ice, and keep
it at room temperature 5 min before injection (seeNote 50).
- For injection, a 27-gauge needle is placed to the syringe.
Remove remaining bubbles before injection. Inject
450–500μL of Matrigel subcutaneously into the flank of the
mice, and leave the needle at this site for 30 s to avoid backflow.
Press a Q-tip on the injection site for 1–2 min until polymeri-
zation starts. Each mouse needs to receive two implants, i.e.,
the control Matrigel and the experimentally treated Matrigel in
the right or left flank, respectively. Make sure that the two plugs
are sufficiently distant from each other.
- Inject the antidote mixture intraperitoneally (final concentra-
tions: 2.5 mg/kg atipamezole, 1.2 mg/kg naloxone, and
0.5 mg/kg flumazenil).
- Leave the mouse for 7 days in the animal facility under normal
housing conditions.
- Sacrifice the mouse by cervical dislocation.
- Take off the plug from each flank by carefully excising it from
the surrounding tissue. Take pictures from plugs for documen-
tation (seeNote 51).
- Transfer the plug into 5 mL of zinc fixative (seeNote 52).
- Incubate the plugs in zinc fixative for 24 h at room
temperature.
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