Tissue preparation was according to established methods44 (link), 45 (link). Briefly, the brain was fresh-frozen and cryosectioned into 20 μm coronal sections that contained LH GFP-labeled MCH neurons. The sections were thaw-mounted onto polyethylene napthalate membrane-coated slides (Leica Microsystems; #11505158; Bannockburn, IL, USA) that had been pre-treated with UV light at 254 nm for 30 min.
After complete dehydration through serial ethanol solutions (75% for 2 seconds, 95% for 10 seconds, 100% for 10 seconds x 2 times), laser microdissection was performed using a Leica LMD6500 system under 40x objective with the guidance of GFP fluorescence. 150 GFP-labeled neurons within the LH/zona incerta region were collected from each rat. Cells were collected into 0.5 ml microtubes (Ambion, Foster City, CA, USA) and lysed in 150 μl of RLT Buffer Plus (Qiagen, Valencia, CA, USA) with 30 sec-vortexing less than 3 hrs after collection, and stored at −80 °C until further processing.
After complete dehydration through serial ethanol solutions (75% for 2 seconds, 95% for 10 seconds, 100% for 10 seconds x 2 times), laser microdissection was performed using a Leica LMD6500 system under 40x objective with the guidance of GFP fluorescence. 150 GFP-labeled neurons within the LH/zona incerta region were collected from each rat. Cells were collected into 0.5 ml microtubes (Ambion, Foster City, CA, USA) and lysed in 150 μl of RLT Buffer Plus (Qiagen, Valencia, CA, USA) with 30 sec-vortexing less than 3 hrs after collection, and stored at −80 °C until further processing.
