Palm microbeam rel 4

All procedures were performed in an RNase-free environment. Animals were sacrificed under deep CO₂-induced anesthesia, brains were quickly dissected in ice-cold PBS, cryopreserved in 20% sucrose overnight, frozen in OCT compound (Polysciences), and stored at −80 °C. Twenty μm thick coronal sections were collected on UV-treated and 0.05% poly-L-lysine coated membrane-covered PEN slides (Zeiss), fixed for 1 min in ice-cold 70% EtOH, incubated for 45 sec in 1% cresyl violet acetate solution (Waldeck) and washed for 1 min each in 70% EtOH and 100% EtOH. Sections were briefly dried on a 37 °C warming plate and immediately processed. The granule cell layer of the DG was isolated by laser capture microdissection using a PALM MicroBeam Rel.4.2 (Zeiss). RNA was isolated from the collected tissue using Rneasy Micro Kit (Qiagen) and reverse transcribed using the SensiFast cDNA Synthesis Kit (Bioline). Quantitative real-time PCR was performed in triplets for each sample using the LightCycler DNA Master SYBR Green I Kit in a LightCycler 480 System (Roche). The relative copy number of Gapdh RNA was used for normalization. Data were analyzed using the comparative CT method (Schmittgen and Livak, 2008 (link)).