Blood and brain samples collection and preparation.
Immediately after the overdose of isoflurane, blood samples, were
obtained by heart puncture and placed in plastic tubes, which were flash
frozen in contact with dry ice. Immediately, after collecting blood
samples, we extracted brains which were also flash frozen in dry ice.
The plasma collection was performed the same day the ELISA assays were
performed. To extract plasma, we first defrosted the blood samples in
contact with ice. Once all the samples were thawed, we kept them 30 min
in the ice. Next, we centrifugated them for 10 minutes at 33500 G, and
we collected the supernatant.
Tissue from amygdala and NAc was collected by punching in brain slices
of 61.2 and 61.6 µm respectively. The bregma coordinates of the brain
slices were from 2.52 to 0.96 mm for NAc, and from -2.04 to -3.24 mm for
amygdala (Figure S2).