- Cryostat (to slice to 4μm; 30 C).
53.þ/þpoly-L-lysine coated glass slides. - Diamond pen for labeling of the glass slides with the tissue slice
treatment condition information. - Lysis solution: 10 mM Tris–HCl, pH 7.4, 160 mM NaCl,
0.05% (v/v) IGEPAL CA-630, 1% (v/v) Triton X-100,
2.5 mM EDTA, 1 mM EGTA, 0.5 mg/mL AEBSF, protease
inhibitor cocktail (seeNote 11). - Ice bucket.
- Microcentrifuge tubes.
- Kit for protein concentration measurement.
3 Methods
3.1 Kidney
Harvesting for Kidney
Slice Preparation (Rats
and Mice) Without
Kidney Perfusion
- This is a two-person procedure from Subheadings3.1–3.3.To
keep the slices in solution as much as possible, two people need
to be present, one to section the other to ensure that slices are
submerged in the buffer. Once the slices are distributed in the
vials under different conditions, then one person can manage
the actions required by the protocol (seeNote 12). - For each condition and time point, there should be an experi-
mental vial and a control vial with no reagent: Prepare a 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 segment of “CO 2 impermeant” capil-
lary tubing through the cap. - In a separate labeled vials (one per condition), place 5–7 mL of
fixative per vial. Keep these vials at RT and near the water bath. - Prior to kidney perfusion and harvest (or harvest alone), pre-
pare all the vials with the CO 2 -equilibrated Ringer buffer at
37 C, place the reagents needed for the incubation, and
continue to bubble the gas through the buffer for all the vials
needed. For example, this is a good time in the protocol to add
the desired AMPK activator (AICAR or A769662) to the vials
(seeNote 13). - Anesthetize the adult male Sprague-Dawley rats with sodium
pentobarbital or with isofluorane. - Harvest the kidneys as in Fig.1.
- Verify each rat is well anesthetized by pinching the tail near the
body. The rat should not react to pain and should have a slow
respiratory rate (Fig.1a). - Lift the abdominal skin with a set of grasping forceps and pull
the skin away from the muscle layer (Fig.1b).
454 Renee Rao et al.