AMPK Methods and Protocols

7. Reagents for protein determination (according to Lowry or
   Bradford).


8. Multichannel pipettor for 100–200μL, sterile reservoir.


9. Sterile tubes.


10. 24-well plates.


11. Cell culture incubator (seeSubheading2.1.1,item 8), incuba-
    tor without CO 2 (37C) or heating plate (37C).

2.1.4 Fixation
and Analysis of Spheroids

1. Phosphate buffer: 200 mM sodium phosphate, pH 7.4. Pre-
   pare stock solutions of 200 mM Na 2 HPO 4 ·2H 2 O (35.6 g in
   1000 mL ddH 2 O, buffer A) and of 200 mM NaH 2 PO 4 ·H 2 O
   (27.6 g in 1000 mL ddH 2 O, buffer B), and mix 770 mL of
   buffer A and 230 mL of buffer B. Check the pH of the solution
   at room temperature. If necessary, adjust the pH to 7.4.


2. PFA stock solution: 4% (w/v) paraformaldehyde (PFA). Weigh
   40 g of paraformaldehyde powder into a glass beaker, and put it
   on a magnetic stirrer in a ventilated hood. Add 400 mL
   ddH 2 O, and heat to 60C while stirring (check temperature!).
   Add 1 N NaOH dropwise to clear the solution. Heat again to
   60 C. Adjust the volume to 500 mL ddH 2 O, and let the
   solution cool down to room temperature. Filter the PFA solu-
   tion into 500 mL of phosphate buffer (pH 7.4) in a 1 L round
   bottom flask. Freeze aliquots. After thawing the solution can
   be stored at 2–8C for up to 1 month.


3. Inverse microscope with software for taking photos.


4. Software for analyzing sprout length and number.


5. Sodium azide: 200 mg/mL sodium azide. Dissolve 200 mg
   sodium azide in 1 mL ddH 2 O, and store at 4C.


6. Parafilm.

2.2 Matrigel Plug
Assay

2.2.1 Generation
of Matrigel Plugs in Mice

1. Mice: Wild-type and AMPKα1 knockout mice as previously
   reported [16, 17].


2. Matrigel, growth factor-reduced, phenol red-free (seeNote
   17 ).


3. PBS: 145 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ,8mM
   Na 2 HPO 4 ·2H 2 O, pH 7.4, autoclaved.


4. Recombinant VEGF 165 protein: Prepare a stock solution of
   10 μg/mL in PBS containing 0.1% (w/v) albumin.


5. Heparin: 12.5 mg/mL stock solution in sterile ddH 2 O.


6. 70% (v/v) ethanol (disinfectant).


7. Anesthetics: 0.1 mg/mL medetomidine, 0.01 mg/mL fenta-
   nyl, 1 mg/mL midazolam.

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