- Place two additional incisions in the abdominal muscle layer
perpendicular to the initial xiphoid to pelvis incision toward
the flanks (Fig.1c). - Using a tissue paper or cotton applicator, displace the intestines
to visualize the right kidney (Fig.1d). - At this point, both kidneys can be harvested and sectioned
(if no perfusion is required, go to Subheading3.3) (Fig.1d).
3.2 Kidney
Harvesting for Kidney
Slice Preparation (Rats
and Mice) with Kidney
Perfusion
- This is also a two-person procedure.
- If you chose to perform kidney perfusion (Fig.1a–d)prior to
the harvestcarefully using a paper tissue or cotton applicator,
dissect away the skin and fascia from the inferior vena cava
(Fig.1e;seeNote 14). - Quickly visualize the diaphragm.
- Ensure that the perfusion pump is functioning and the perfu-
sion tubing is free of air and the buffer container is at 37C and
equilibrated with the CO 2 /air mix prior to stating the
experiment. - Ensure that at the start of the perfusion, the buffer 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. You may need to place two
flank incisions to open the rib cage upward. - Place the needle in the left ventricle (if obtaining slices from
mice, use a smaller needle;seeSubheading2). - Ask an assistant to increase the pump speed to 4–5 mL/min.
- Once the kidney and the inferior vena cava appear expanded,
place a small puncture on the inferior vena cava and perfuse the
kidney until it becomes pale, using the suction to keep the field
clean for continued observation. - After the kidneys are well perfused, immediately harvest both
kidneys and place them in a petri dish.
3.3 Kidney Slice
Preparation
- Ensure that the Stadie-Riggs microtome and blade are ready
(Fig.2). - Quickly with a scalpel or blade, cut the kidney into ~5 mm
sections transversally and radially (each thick section should
have some papilla). - Place these thicker kidney sections in a beaker with Ringer
buffer at 37C equilibrated with the CO 2.
456 Renee Rao et al.