- Placing the kidney slices once for 15 min in quenching buffer
(quench step). - Rinse tissue again three times, each time for 15 min in 1PBS
at RT (seeNotes 16and 17 ). - The kidney slices can now be stored at 4C in PBS azide and
can now be processed for paraffin sections and histology. - For immunolabeling, proceed by placing tissue in 30% sucrose
overnight at 4C or at RT for a few hours until it floats. - Place the slice onto a plastic cryomold and flatten it to the
bottom of the mold. Freeze in the cryostat chamber at
30 C. Mount onto the cryostat chuck and section (usually
4 μm thickness) (seeNote 18). For immunolabeling,seeref.
11–13 (Fig.4).
3.5 Kidney Slice
Frozen Tissue Lysis
Preparation
- Obtain frozen tissue from 80 C freezer and transport on ice
(seeNote 19). - Incubate on ice in cold room for ~30 min until thawed and/or
soft enough to homogenize. - Put necessary tools in cold room to chill (there must be suffi-
cient tubes for all samples).
Fig. 4Confocal images of tissue slices. Kidney slices from rats (a) and mouse (b) kidney immunolabeled for
transport proteins aquaporin-2 (AQP2) and epithelial sodium channel (ENaC). The slices were treated with an
AMPK activator (AICAR) in the absence or presence of a vasopressin (a) or untreated (b). After fixation and
cryopreservation, slices were immunolabeled with antibodies against the different transport proteins and
imaged using a confocal microscope
AMPK Studies using Kidney Slices 459