Arturusxt micro dissection system

Mice were euthanized by overdose with isoflurane and the brains were quickly dissected out and frozen at −80 °C. The frozen mouse brains were then sectioned with a cryostat at −20 °C and loaded onto the PAN membrane frame slide (Applied Biosystems, Foster City, CA). The mDANs were dissected by the LCM with the ArturusXT micro-dissection system (Applied Biosystems) based on the presence of green fluorescent proteins (GFP) as described previously⁷ . Briefly, the total RNA was extracted from around 800 isolated mDANs per mouse brain with the PicoPure Isolation kit (Applied Biosystems) and the cDNAs were synthesized with the First Strand kit (QIAGEN, Valencia, CA) from equal amounts of total RNAs in the comparison groups. The SYBR Green real-time PCR detection method was used to quantitate the calcium channel expression. All the primers used in the qPCR were from QIAGEN and tested by the manufacturer. The expression levels were calculated as fold changes normalized with the β-actin. Data were statistically compared with unpaired t test, two tailed.

Mouse brains of different genotypes and treatments were quickly dissected out and frozen down at −80 °C. The frozen mouse brains were sectioned with a cryostat at −20 °C and loaded onto the PAN membrane frame slide (Applied Biosystems, Foster City, CA). The dorsal striatum was dissected by the LCM with the ArturusXT micro-dissection system (Applied Biosystems) based on the anatomic landmarks in the striatum such as corpus callosum (CC), lateral ventricle (LV) and nucleus accumbens (Acb). The total RNAs were extracted with the PicoPure Isolation kit (Applied Biosystems) and the cDNAs were synthesized with the First Strand kit (QIAGEN, Valencia, CA) from equal amounts of total RNAs in the comparison groups. The SYBR Green real-time PCR detection method was used to quantify the Aldh1a1 and opioid receptors as well as the peptides. All the primers used in the qPCR were from QIAGEN and tested by the manufacturer. Gene expression was calculated as fold change normalized to β-actin.