3.2 Dissection
of the Hypothalamus
and Protein Extraction
Carry out all procedures in ice otherwise specified.
The whole hypothalamus can be dissected in fresh tissue with a
precision scissors. For nucleus-specific dissection, the brain needs
to be frozen.- Immediately after sacrifice, position the brain with the hypo-
thalamus upward and the cerebral hemispheres downward in
foil, over dry ice. Allow the tissue to completely freeze before
closing the foil to stock at 80 C. - Before dissection, let the brain half-thaw in ice and put it in the
brain matrix with the hypothalamus upward. Rinse it with few
drops of PBS and position a razor blade at the start of median
eminence (ME; for the dissection of hypothalamic nuclei).
Position the other razor blade at 2 mm of the first one. Cut
the brain and position the slice in a plastic plate. With a surgical
blade n15, dissect the area of the specific nuclei with the help
of a Brain Atlas [68](seeFig. 1). Immediately introduce the
tissue in a 2 mL plastic tube with a stainless steel grinding bead,
add the appropriate BLYSpiquantity (seeNote 7), and disrupt
it in the bead mill homogenizer for 2 min at 30 Hz (1800
oscillations/min) (seeNote 8). Keep the homogenate always in
ice. Collect it to a new plastic tube and centrifuge for 30 min at
13,200 rpm (16100g)at4C(seeNote 9). Collect the
supernatant and transfer to a clean plastic tube. - Determine protein concentration with the traditional Bradford
assay reagent. Prepare BSA standard curve as follows: Prepare a
1 mg/mL BSA standard by diluting 1 mg BSA in 1 mL of
distilled water. Using 1 mg/mL standard, prepare standard
Fig. 1Specific dissection of hypothalamic nuclei. (a) Position of rat brain in the acrylic brain matrix. Black lines
are signaling the position of razor blades for delimitation of hypothalamic region containing ARC, VMH, and
LHA. (b) Coronal section of rat brain showing the localization of the nuclei.3Vthird ventricle,ARCarcuate
nucleus of the hypothalamus,LHAlateral hypothalamic area,MEmedian eminence,VMHventromedial
nucleus of the hypothalamus
Methods for Brain AMPK 441