Sc 52 g

Mice were tested with the self-stimulation protocol described above in the presence of food for 2.5. Immediately after the self-stimulation experiment, each mouse was perfused transcardially with PBS buffer followed by formalin. Brains were removed, postfixed in 4% PFA and transferred to PBS buffered 20% sucrose. Free floating sections (40 µm) were prepared with a cryostat, blocked (3% BSA, 2% NGS, and 0.1% Triton-X in PBS for 2 hr), and then incubated with primary antibody (chicken anti-GFP, Abcam, ab13970, 1:1000; goat anti-Fos, Santa Cruz, SC52G, 1:500) overnight at 4°C. Samples were washed, incubated with secondary antibody (goat anti-chicken Alexa 488 secondary antibody; Invitrogen, 1:1000; donkey anti-goat Alexa 568 secondary antibody; Invitrogen, 1:1000) for 2 hr at room temperature, washed, mounted, and imaged with a confocal microscope. Images for direct comparison are imaged with the same settings.
To quantify the percentage of AgRP neurons that express Fos, we first identified 100 putative AgRP cells from each mouse based on GFP fluorescence using the ImageJ Cell Counter Plugin. We then manually quantified the presence or absence of Fos staining in those previously defined cells.

Mice were anesthetized with phenytoin/pentobarbital and perfused with phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH = 7.4). After an overnight post-fix in 4% PFA, brains were cryoprotected overnight in 30% sucrose before being embedded in OCT and stored at −80 °C. Free-floating sections (30 µm) were prepared with a cryostat. They underwent three washes in PBST (PBS + 0.3% Triton X-100) and were blocked in 3% normal donkey serum in PBST (1–2 h). Sections were then incubated overnight at 4 °C in primary antibodies, including rabbit anti-Satb2 1:2500 (ab34735, Abcam), mouse anti-Satb2 1:1500 (ab34735, Abcam), rabbit anti-Fos 1:2000 (#22505, Cell Signaling; ab190289, Abcam), goat anti-Fos 1:300 (sc-52-G, Santa Cruz), chicken anti-GFP 1:10,000 (ab13970, Abcam), goat anti-CGRP 1:1000 (ab36001, Abcam), and/or rabbit anti-dsRed 1:1000 (632475, Takara). The next day, the tissue was washed three times in PBS and incubated 1–2 h in appropriate secondary antibodies including Alexa Fluor 488/594 donkey anti-sheep, Alexa Fluor 488/594 donkey anti-goat, Alexa Fluor 488/594 donkey anti-rabbit, or Alexa Fluor 488 donkey anti-chicken (1:500; Jackson Immunoresearch Laboratories). Tissue was washed three times in PBS, mounted onto glass slides, and coverslipped with Fluoromount-G (Southern Biotech).