Lmd6500 system

Tissue preparation was according to established methods^{44 (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.

Laser microdissection was performed using a Leica LMD6500 system (40X objective in the fluorescence mode for SST neurons or bright-field mode for pyramidal neurons). Cutting and collection steps were subsequently examined in fluorescent or bright-field mode. 100 cells from the mouse cingulate cortex per animal were collected in 0.5 ml microtube caps (Ambion, Foster City, CA, USA) and lysed by vortexing for 30 sec in 150 μl of RLT Buffer Plus (Qiagen, Valencia, CA, USA) within a 3-hour time span, and stored at 80 °C until further processing. Total RNA was then extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s instructions. RNA samples were amplified with NuGEN® Ovation Pico WTA System V2 (NuGen, San Carlos, CA). The fragmented labeled cDNA samples were processed and hybridized to Affymetrix® Mouse Gene 1.1 ST Array Plates (Affymetrix). Gene expression information was obtained with the Affymetrix console software. Genes showing significant differences (Student t-test p<0.005; >1.2 fold cutoff) in expression between SST neurons from stressed and unstressed mice were analyzed using the IPA software (Ingenuity Systems, Redwood, CA).