- Using scissors, make an incision in this ventral skin from the
direction caput to cauda along the central abdominal line
(xiphoid to pelvic area) (Fig.1b). - Expose the abdominal muscle layer by placing two additional
incisions on the skin perpendicular to the first cut toward each
flank taking care to avoid major blood vessels (Fig.1b). - Using the same grasping forceps, pull the muscle away from
major intra-abdominal organs (Fig.1c). - Place a small incision in the middle of the abdominal muscle
layer, and by carefully visualizing that the scissors are not
perforating any internal organs, cut the muscle along the
linea alba toward the xiphoid and the pelvis. Avoid touching
the diaphragm and the bladder (Fig.1c).
Fig. 1Diagram of the tissue-harvesting surgery. The depicted procedure relates to Subheading3.1 (without
kidney perfusion) (a–d) and Subheading3.2 (with kidney perfusion) (a–e). (a) Verify each rat is well
anesthetized by pinching the tail (arrow: position tail pinch). The rat should not react to pain and should
have a slow respiratory rate. (b) Expose the abdominal muscle layer by placing two additional incisions on the
skin perpendicular to the first cut toward each flank taking care to avoid major blood vessels. Using the same
grasping forceps, lift the muscle layer away from major intra-abdominal organs underneath (gray arrow). (c)
Place a small incision in the middle of the abdominal muscle layer, and by carefully visualizing that the
scissors are not perforating any internal organs, cut the muscle along the linea alba toward the xiphoid and the
pelvis. Avoid touching the diaphragm and the bladder. Place two additional incisions in the abdominal muscle
layer perpendicular to the initial xiphoid to pelvis incision toward the flanks. (d) Using a paper tissue or cotton
applicator (depicted), displace the intestines to visualize the right kidney. At this point, both kidneys can be
harvested and sectioned (if no perfusion is required, e.g., go to Subheading3.3 for instructions to prepare
kidney slices). (e) Carefully using a paper tissue or cotton applicator, dissect away the skin and fascia from the
inferior vena cava. Quickly visualize the diaphragm (black dotted line). Ensure that the perfusion pump is
functioning and the perfusion tubing is pumping slowly (one drop every 2–3 s) and that the suction tubing is
functional and the vacuum suction is on. With scissors, place an incision in the rightmost aspect of the
diaphragm and quickly dissect it frontally toward the left flank with care not to puncture the heart (black arrow
and scissors, cut along the gray dashed line). You may need to place two flank incisions to open the rib cage
upward. Place the needle in the left ventricle (red arrow). Ask an assistant to increase the pump speed to
4 mL/min. Once the kidneys and the inferior vena cava appear expanded, place a small puncture (purple arrow
and scissors) on the inferior vena cava and perfuse the kidney until it becomes pale, using the suction to keep
the field clean. When kidneys appear pale, harvest immediately
AMPK Studies using Kidney Slices 455