Computer imaging device

Brain tissues from the ipsilateral SN were dissected at the indicated time points after LPS or PBS injection with capsaicin or vehicle, and total RNA was extracted in a single step using RNAzol B (Tel-Test, Friendswood, TX) following the instructions of the manufacturer. Total RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Promega, Madison, WI) and random primers (Promega). The primer sequences used in this study were as follows: 5′-TGATGTTCCCATTAGACAGC-3′ (forward) and 5′-GAGGTGCTGATGTAC CAG TT-3′ (reverse) for IL-1β (378 bp); 5′-ACACTCTATCACTGG CATCC-3′ (forward) and 5′-AAGGGACACCCTTTCACAT-3′ (reverse) for COX-2; 5′-GCAGAA TGTGACCATCATGG-3′ (forward) and 5′-ACAACCTTGGTGTTGAAGGC-3′ (reverse) for iNOS (557 bp); and 5′-TCCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-AGATCCACAACGGATACATT-3′ (reverse) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 308 bp). The PCR amplification consisted of 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s (for IL-1β and GAPDH or 54 °C for 30 s for iNOS) and extension at 72 °C for 90 s. PCR products were separated by electrophoresis on 1.5% agarose gels, after which the gels were stained with ethidium bromide and photographed. For semiquantitative analyses, the photographs were scanned using a Computer Imaging Device and the accompanying software (Fujifilm, Tokyo, Japan).

Brain tissues for Western blotting were prepared as previously described [16 (link), 21 (link)]. Briefly, the mice hippocampal tissues were homogenized and centrifuged at 4°C for 20 min at 14,000 g. The supernatant was transferred to a fresh tube and the concentration was determined using a BCA kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins analyzed on gel electrophoresis were transferred to polyvinylidene difluoride membranes (Millipore) using an electrophoretic transfer system (Bio-Rad Laboratories), and then the membranes were incubated overnight at 4°C with specific primary antibodies: anti-β-actin (1 : 4000, Cell Signaling), anti-LC3B (1 : 1000, Cell Signaling), and anti-TNFα (1 : 1000, R&D system). After washing, the membranes were incubated with secondary antibodies (Amersham Biosciences, Piscataway, NJ), and the blots were finally developed with the ECL Western blotting detection reagents (Amersham Biosciences). For semiquantitative analyses, the density of the immunoblot bands was measured with the Computer Imaging Device and accompanying software (Fuji Film, Tokyo, Japan).