AMPK Methods and Protocols

8. Antidotes to anesthetics: 5 mg/mL atipamezole, 2.4 mg/mL
   naloxone, 1 mg/mL flumazenil.


9. 1 mL syringes, 20- and 27-gauge needles.


10. 2 mL Eppendorf tubes, 15 mL Falcon tubes, pipette tips.


11. Scissors, scalpel.


12. Zinc fixative (seeNote 18).

2.2.2 Embedding
of Matrigel Plugs in Paraffin
and Sectioning

1. Tissue cassettes.


2. Spin tissue processor (Histokinette).


3. Ethanol (99%, 96%, 70% (v/v)).


4. Xylene.


5. Ethanol:xylene: 2:1 and 1:2.


6. Paraffin.


7. Modular tissue embedding center.


8. Microtome.


9. Paintbrush, 20- and 27-gauge needles.


10. Water bath at room temperature and 37C.


11. Polylysine-coated microscope slides.

2.2.3 Staining of Matrigel
Plugs (CD31
Immunofluorescence)

In addition toitems 3and 4 in Subheading2.2.2. above, the

following material is required:

1. Ethanol (50% (v/v)).


2. Citrate buffer: 0.01 M Na 3 C 6 H 5 O 7 ·2H 2 O, pH 6.0.


3. Microwave.


4. PBST: PBS including 0.01% (v/v) Triton X-100.


5. Immunostaining system.


6. Blocking solution: PBST, 10% (v/v) serum (seeNote 19),
   0.25% (w/v) bovine serum albumin-c (BSA-c) (seeNote 20).


7. Anti-CD31 antibody applicable on paraffin sections (seeNote
   21 ).


8. Secondary antibody coupled to a fluorescent dye (seeNote 22).


9. Fluoromount-G aqueous mounting medium.


10. Coverslips (0.17 mm thickness).


11. Laser scanning microscope or epifluorescent microscope with
    software including stitching function.


12. ImageJ (NIH) or comparable analyzing software.

524 Katrin Spengler et al.