Gel imaging analysis system

After spatial memory evaluation, rats (n = 3) in each group were sacrificed, and brains were removed. Rat hippocampus were chopped into small pieces, homogenized in 0.5 ml of RIPA buffer. The dissolved proteins were collected from the supernatant after centrifugation at 12 000 × g at 4°C for 5 min. The supernatant was collected for Western blotting analysis. The protein concentration of the supernatant were determined with the BCA Protein Assay reagent kit following the manufacturer’s protocols. Equal amounts of protein samples were separated by SDS/PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 2 h and incubated with rabbit anti-5-LO (1: 300; Beijing Boaosen Biotechnology Co., Ltd., Beijing, China) and rabbit anti-β-actin polyclonal antibody (1: 1 000; Beijing Boaosen Biotech-nology Co., Ltd., Beijing, China) at 4°C overnight. After washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000) for 2 h at 37°C. Finally, the membranes were reacted with the ECL reagents and exposed on an X-ray film. The optical densities of 5-LO and β-actin bands on the X-ray film was measured using a Bio-Rad gel imaging analysis system (Bio-Rad Laboratories). The results were expressed as 5-LO/β-actin ratios.

Total RNA was extracted from rat primary neurons according to RNAiso Plus reagent (Takara, China), cDNA templates was synthesized by PrimeScript^® 1st Strand cDNA Synthesis Kit (Takara, China) following the manufacturer's instructions, and then amplified by using the Premix PCR kit (Cwbio, China). PCR products were separated by 2% agarose gel electrophoresis. All the samples were normalized by the expression level of β-actin. The optical density (OD) values were measured with a Bio-Rad gel imaging analysis system (Bio-Rad, USA). According to GenBank mPGES-1, EP1, EP2, EP3, EP4 mRNA sequence of rats and design primers. Target genes and reference gene (β-actin) primers by the Sangon biotech design and synthesis, primer sequences are listed in Table 1.

The paraffin sections (the same as those for H&E staining) were used for immunohistochemical assays following the manufacturer’s protocol: The sections were deparaffinized in xylene and hydrated through a series of graded ethanol, and washed thrice with PBS (0.02 mmol/L, pH 7.4) for 3 min. The sections were incubated with 3% H₂O₂ for 10 min. After the section was rinsed for 3 min × 3 with PBS, the rehabilitation of antigen was performed by microwave oven. Then the sections were incubated with a polyclonal antibody to NF-κBp65, (diluted 1:100) overnight at 4°C. The sections were rinsed for 3 min × 3 with PBS before incubation with biotinylated goat anti-rabbit IgG antibody for NF-κBp65, for 40 min at 37°C, and incubated with streptavidin for 20 min,and then rinsed for another 3 min × 3 with PBS before reaction with DAB solution. The sections were counterstained with hematoxylin and then observed under a microscope. The absorbance value of 5-LO and β-actin mRNA was measured using a Bio-Rad gel imaging analysis system (Bio-Rad Laboratories). The absorbance ratio of 5-LO mRNA to β-actin mRNA was defined as the 5-LO mRNA relative quantity.