Table 2.

Step-by-step protocol for the isolation and perfusion of the right mouse adrenal gland

1	Sedate the mouse with isoflurane in a small dedicated gas chamber and euthanize via decapitation
2	Swiftly open the abdominal cavity to the mouse’s right side in a big D-shaped window
3	Place 2 ligatures in position around the aorta, ligatures I and II (figure). Ligature I, distal to the left renal artery, ligature II cranial to the left renal artery, distal to the right renal artery. (Make all ligatures, except for ligature VI, using square knots)
4	Make an incision in the aorta, distal to ligature I, and insert a catheter of similar diameter to the aorta with ringer solution flowing at around 1.3 mL/min
5	Move the catheter up inside the artery to pass ligature II, without surpassing the right renal artery, and close the ligatures to keep the catheter in place (the extra threads from each ligature will be used further in the procedure so, do not cut them out)
6	Make an incision on the cava distal to where ligature I is placed in the aorta
7	Place a third ligature (Fig. 2D, ligature III) around the aorta, distal to the celiac trunk, but cranial to the right renal artery. Close this ligature as soon as in position (the artery should be translucid and free from red blood cells at this point)
8	Place and close a fourth ligature (figure, ligature IV) around the superior mesenteric artery, in the vicinity of the aorta (at this point, the right kidney and the right adrenal gland are perfused, and the kidney should become pale)
9	Proceed by opening the chest cavity in V-shape from the xiphoid cartilage separating the ventral segments of ribs from both sides of the sternum. Cut the IVC near the diaphragm
10	Place, but do not close yet, ligature V around the IVC distal to the right renal vein but cranial to the left renal vein as illustrated; put special care not to damage the right renal artery nor the aorta
11	Cut out the preparation and transfer it to a small custom-made chamber where adequate temperature and moisture can be maintained • Cut the mesenteric artery and the left renal vein and artery, separating the left kidney and the intestines from our area of interest • Set apart the liver and gall bladder from the diaphragm • Hold all the extra threads from the ligatures in a bundle • Cautiously pull the threads bundle to help guide the preparation out of the abdominal cavity, and make an incision cutting the cava, aorta, and muscles distal to the aorta catheter • Carefully cut out the right kidney/adrenal gland/liver by cutting muscles as close to the spine as possible up to the diaphragm • Finally cut the diaphragm and the aorta above the liver • Cut and discard around 90% of the left liver lobe which will be hanging at the bottom, leaving behind the rest of the lobes • Transfer the preparation to the custom-made incubation chamber
12	Locate the rest of the left liver lobe and cut it out together with the caudal liver lobe, exposing the IVC
13	Preposition ligature VI around the liver and prepare a surgeon knot to later close the ligature. Let it wide open for now
14	Insert the collecting catheter, a PE tubing with a flange at the opening made by briefly presenting it to fire, into the IVC via the liver, and gently guide it up to close to the right renal vein
15	Close tightly ligature VI around the liver, the muscle bundles of the diaphragmatic crura, and the catheter, making sure that it runs between the right and the median lobes
16	Retract the collecting catheter until it the flange reaches the ligature (will stop it in position)
17	Close tightly ligature V, cut out the extra threads, and close the chamber
18	Start of the experimental protocol