- For mice preparations, use lighter pressure or the kidney tissue,
and/or a thinner cover (based on the original cover depicted in
Fig. 2d) can be built to modify the Stadie-Riggs tissue slicer. - AMPK activator: 5-Amino-1-β-D-ribofuranosyl-imidazole-4-
carboxamide (AICAR). Use at a final concentration of
1–2 mM. - AMPK activator: 200μM A769662 (seeNote 9).
- Glass scintillation vials for incubation of the slices. Label one
vial per incubation condition. The vial must have a perforated
cap through which to feed a “capillary” tubing through
the cap. - At least two beakers with CO 2 -equilibrated Ringer buffer to
hold slices until they are placed in the treatment vials. - Set up a system of manifolds for gas delivery to the Ringer
buffer solution (stock and for each of the slice incubation
conditions). Use CO 2 impermeant tubing. - Water bath with holders for the scintillation vials. Set the bath
at 37C. - Gas tank with appropriate regulator (5% CO 2 /balance air).
- 1phosphate-buffered saline (PBS) without calcium and mag-
nesium: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ,
8mMNa 2 HPO 4 ·7H 2 O, pH 7.4. Check pH prior to use.
Prepare on the day of the experiment and keep at RT. - Fixative for slices: 4% (w/v) paraformaldehyde in 1 PBS
without calcium and magnesium (seeNote 10). - Liquid nitrogen container with liquid nitrogen ready at the
start of the experiment. - Prepare at least one microcentrifuge tube per slice incubation
condition for rapid freezing (with one venting hole in the
cover). Storage of frozen slices at 80 C. - Quenching buffer: 0.2 M NH 4 Cl in PBS. Add 2.1 g dry
ammonium chloride (NH 4 Cl) to 500 mL glass beaker. Add
1 PBS to 200 mL and use a stir bar to dissolve the ammonium
chloride at RT. Quench buffer can be stored at RT. - Cryoprotectant solution: 30% (w/v) sucrose, 0.02% (w/v)
sodium azide in PBS. Prepare several days before the experi-
ment and keep at 4C. Prepare 1 L of 1PBS with 0.02%
sodium azide. Weigh 300 grams of sucrose and combine with
~800 mL of 1PBS with 0.02% sodium azide. Use a stir bar to
slowly dissolve the sucrose in solution at room temperature.
After the sucrose is completely dissolved, bring the solution to
a total volume of 1 L by adding more 1PBS with 0.02%
sodium azide. - OCT from Tissue-Tek (cryo-embedding material).
AMPK Studies using Kidney Slices 453