AMPK Methods and Protocols

quickly activated if samples are not properly handled, many

researchers harvest the kidney following specific protocols to pre-

vent non-specific AMPK activation. For example, the recom-

mended harvesting protocol for the kidney to prevent

non-specific AMPK activation involves freezing the kidney in situ

while the animal is under anesthesia [16].

The purpose of this chapter/protocol is to document the kid-

ney slice technique and the tissue preparation for immunolabeling

and biochemistry preparations. However, we do not describe the

specifics of the latter techniques beyond the tissue lysis and fixation

steps.

2 Materials

1. 300 g male Sprague-Dawley rats matched in age (seeNote 1for
   rat selection). All protocols should be approved by the appro-
   priate institutional regulatory committee at the appropriate
   institution.


2. 25 g mice (seeNote 2for mouse selection).


3. Millipore purified water or equivalent to prepare all buffers.


4. Anesthetic agent: pentobarbital sodium i.p. (seeNote 3).


5. Alternative anesthetic agent: isoflurane to be used with vapor-
   izer machine (seeNote 4).


6. Solution stock A: 2.4 M NaCl. Weigh 70.13 g sodium chloride,
   then transfer to 1 L graduated cylinder, and add pure water to
   final volume of 500 mL. Mix until dissolved at RT and store at
   4 C until needed.


7. Solution stock B: 0.5 M NaHCO 3. Weigh 21.003 g of
   NaHCO 3 , then transfer to 1 L graduated cylinder, and add
   pure water to final volume of 500 mL. Mix until dissolved at
   RT and store at 4C until needed.


8. Solution stock C: 66.6 mM KH 2 PO 4 ,16mMK 2 HPO 4. Weigh
   4.532 g potassium phosphate monobasic (KH 2 PO 4 ) and
   1.393 g potassium phosphate dibasic (K 2 HPO 4 ), then transfer
   both to 1 L graduated cylinder or glass beaker, and add pure
   water to final volume of 500 mL. Mix at RT until dissolved and
   store at 4C until needed.


9. Solution stock D: 24 mM CaCl 2 , 24 mM MgCl 2. Weigh
   1.332 g calcium chloride anhydrous (CaCl 2 ) and 2.440 g mag-
   nesium chloride hexahydrate (MgCl 2 ), then transfer to 1 L
   graduated cylinder or glass beaker, and add water to final
   volume of 500 mL. Mix at RT until dissolved and store at
   4 C until needed.
   10.D-Glucose.

AMPK Studies using Kidney Slices 451