- Place one of these thick sections on the Stadie-Riggs slicer
(Fig. 3a–h) and quickly obtain a thin kidney tissue slice
(~0.5 mm in thickness). - Retrieve the thin kidney slice quickly to place it in a second
beaker with CO 2 -equilibrated Ringer at 37C. - Continue to obtain slices and place them in the beaker with the
other slices. - The entire procedure of sectioning both kidneys from the same
animal should be performed in less than 5 min. - At the end of the sectioning, incubate all the thin kidney slices
for 15 min in equilibrated Ringer solution before beginning
treatment. - Place the slices into the vials for the treatments (Fig.3h).
- For AICAR and A769662 treatment, we suggest a minimum of
75-min incubation prior to fixation or rapid freezing. - At the end of the incubation time, rapidly place the slices in
either fixative or in a microcentrifuge tube that must be rapidly
submerged in liquid nitrogen.
3.4 Kidney Slice
Sectioning
for Immunolabeling or
Histology
- After the above treatments, always keep the slices for each
treatment in a separate appropriately labeled container, and
place the slices in fixative (4% paraformaldehyde in PBS).
Fig. 2Stadie-Riggs tissue slicer (microtome) parts and assembly. (a) Place a blade (new for each experiment)
on the handle using a needle holder or hemostat. The blades are extremely sharp and should not be handled
directly with hands (b–d). To assemble the slicer, the three parts must fit Stadie-Riggs slicer. Cap the slicer.
Tighten knobs until kidney section is held firmly in place leaving enough space for the blade to fit. Usually the
long piece of the microtome is held with the left hand and the blade with the right hand (seeFig. 3)
AMPK Studies using Kidney Slices 457