Ribonuclease rnase inhibitor

Trizol reagent (T9424), chloroform (C2432), and isopropanol (I9516) were purchased from Sigma-Aldrich (St Louis, MO). Taqman PCR core kit (N8080228), MuLV Reverse Transcriptase (N8080018), and Ribonuclease (RNase) Inhibitor (N8080119) were obtained from Life Technologies (Grand Island, NY). RNase free micro tubes and pipette tips were used in RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR).

LCM was performed on two membrane slides per animal as previously described (78 (link)). In brief, brain tissue sections were placed on a cold block, fixed for 2 min in ice-cold 75% ethanol solution, and then rinsed thrice with ice-cold nuclease-free PBS (pH 7.4, Life Technologies). Brain sections were stained in a 1:220 dilution of goat anti-rat IgG + 500 U of ribonuclease (RNase) inhibitor (Life Technologies). After 1-hour exposure, sections were rinsed three times with nuclease-free PBS and dehydrated sequentially with 75, 95, and 100% ethanol for 15 s each. Sections were placed on the ice block to dry for 5 min before LCM using a laser microdissection microscope (Leica).