Palm robot microbeam system

We extracted RNA from the frozen midbrain of ~30 healthy controls obtained from the Oxford MRC Control Brain Bank. South Central–Oxford C Research Ethics Committee approved the study (ethical license REC15/SC/0639). The RNA integrity number (RIN) was analyzed in these samples using Bioanalyzer (Agilent Technologies, Santa Clara, CA) and ranged between 6.6 and 9.2. Only 7 healthy controls with the highest RIN scores (≥8) were selected for the further analysis. The mean age at death in these 7 healthy controls (2 female, 5 male) was 70.7 ± 12.3 years (range = 56–93 years). Briefly, 10μm‐thick sections of the frozen midbrain were cut at the level of the 3rd nerve, dehydrated in ethanol series, and stained with cresyl violet in strictly RNA‐free conditions. Single neuromelanized neurons were isolated from separate, nonoverlapping ventral and dorsal tiers of SNpc using anatomic criteria and harvested from the stained cryosections by using a PALM Robot–Microbeam system (Carl Zeiss, Oberkochen, Germany; Fig 1).

Seven days after surgery, rats were decapitated after lethal intraperitoneal injection of pentobarbital sodium (100 mg/kg). L4/L5 DRGs were rapidly dissected (<10 min) in RNAse free environment, immediately embedded in O.C.T. compound (Sakura Finetek), placed at −20 °C for 1 h and then stored at −80 °C until cryosection. The cryosection was effectuated the same day of the microdissection. DRG tissue was sectioned at 10 μm and mounted on PALM POL-covered membrane slides (Zeiss). LCM was performed immediately after the sectioning using the Palm Robot-Microbeam system (Zeiss, Suppl. Video 1). Duration of LCM was limited to a maximum of 25 minutes per slide to preserve the RNA integrity. Samples were collected into microfuge caps moistened with a drop of mineral oil (Sigma-Aldrich), covered with 15 μl of Qiagen RTL buffer (Qiagen) with 1% β-mercaptoethanol (Sigma-Aldrich) and stored at −80 °C.