- Do not microwave “sugar” containing solutions as the micro-
wave process may “caramelize” the solution. - This procedure is optional, as some of the transporter proteins
may change their subcellular localization upon kidney perfu-
sion via the left ventricle. However, this procedure helps keep
tubules open after sectioning and washes the vasculature of the
kidney to remove hormones that may affect intracellular pro-
tein traffic. - The first and second washes will have high concentrations of
paraformaldehyde and should be properly discarded as chemi-
cal waste. All other rinses can be disposed of as normal liquid
waste. - The tissue should be processed at room temperature, but
samples should not be allowed to sit out longer than necessary.
Perform all steps for the times indicated. Do not allow rinse
steps to exceed 20 min unless the tissue is unusually thick. - For a particularly helpful setup, place a small funnel over the
spout of an empty bottle (i.e., an empty media bottle). Line the
funnel with a clean tissue paper. Pour solutions into the tissue
paper, using a Pasteur pipet, to direct flow and prevent the
tissue from falling. If any tissue does fall out, it will catch in the
tissue paper and can be retrieved with forceps. - Cryostat use tips: These are not necessarily part of the protocol
but are good introductory advice while handling the kidney
slices. Be careful with the blade; it is very sharp. It is recom-
mended to change the blade before each sectioning. Be espe-
cially careful not to damage the glass of the anti-roll plate with
the metal part of the brushes. Use brushes without metal,
which often chinks the glass plate. Be sure to judge the distance
of the anti-roll plate to the blade; it has to be just right to
prevent rolling without jamming the tissue. When leaving the
cryostat unattended, place a thin layer of OCT to protect the
tissue block; lock handle to prevent tissue damage; when fin-
ished sectioning, place the tissue back in cryomold, wrapped in
aluminum foil, labeled, and placed at 20 C for later use. - There are many different methodologies/protocols to gener-
ate a kidney homogenate. Our protocol of immediately freez-
ing the kidney tissue and then processing at a later time is done
solely for the purpose of ensuring that AMPK activity matches
the level of AMPK activation that would be expected from the
condition. If needed, side-by-side comparisons of homogeni-
zation and immunoblot protocols should be performed. It is
an important point that all tissues being examined for AMPK
downstream effects must be frozen right after the incubation in
the Ringer buffer. If tissue is left on ice for lysis at a later time,
462 Renee Rao et al.