Benzidine (DAB) as a chromogen and Hematoxylin as a counter stain

Benzidine (DAB) as a chromogen and Hematoxylin as a counter stain (Vector, Burlingame, CA) by following the suggested manufacturer's protocol. Specifically, paraformaldehydefixed and paraffin-embedded slide-mount tissue sections using 3 brains from each group were heated in boiling citrate-based antigen unmasking solution for 1 min for better antigen retrieval by the primary antibodies. Section slices were then incubated overnight with primary antibody in PBS containing 2 BSA and 2 normal goat serum followed by secondary antibodies coupled to horse radish peroxidase. The numbers of stained cells in the 4 well stained high magnification fields (400x), which were taken by using a H550L microscope and DS-Fi1c digital camera (Nikon, Tokyo, Japan), were counted using i-Solution (ver. 7.5; IMT i-Solution, Coquitlam, BC, Canada), an image analysis software. Sections stained with anti-neurofilament antibody were used to measure the length of neurite projections using ImageJ software (NIH). Length data were statistically compared by Student's t-test.Composition and concentration of fatty acids in the brain and perilla0.25 m (J W Scientific, Folsom, CA)). Fatty acid peaks were determined based on the retention times of the FAME standards. The area of externally added 17:0 standard was used to calculate the concentration of fatty acids by proportional comparison of the peak areas. All samples were run five times, and the values were averaged for all subsequent analyses.Additional filesAdditional file 1: Table S1. Fatty acid composition in the brain of rats fed control or perilla diets for 3 weeks or 3 months. Additional file 2: Table S2. 20 major proteins in the brain of rats fed perilla diets. Additional file 3: Table S3. Nutrient compositions of the diets. Competing interests The authors declare that they have no competing interests. Authors' contributions JL and SP contributed to the overall conception and design of the project, carried out all the experiments, analysis and interpretation of the data, performed mass spectrometry analysis, and preparation of the manuscript. JYL carried out animal behavioral tests, mass spectrometric analysis and interpreting mass spectrometry data. YKY contributed with critical discussions on fatty acid analysis, fatty acid metabolism in brain and data interpretation in fatty acids. JSK contributed to the animal diet formulation, design of animal behavioral test, data analysis and interpretation. The corresponding author JL conceived the study, and participated in its design and coordination, data analysis and interpretation, and preparation of the manuscript. All authors read and approved the final manuscript. Acknowledgements This research was supported by the grant (#2009-0077553) from National Research foundation of Korea (NRF). The authors are indebted to Dr. Kilsoo Kim for his advice in rat brain anatomy. Received: 17 August 2012 Accepted: 29 November 2012 Published: 5 December 2012 References 1. Goelet P, Castellucci VF, Schacher S, Kandel ER: The long and the short of long-term memory molecular framework. Nature 1986, 322:419?22. 2. Costa-Mattioli M, Sossin WS, Klann E, Sonenberg N: Translational control of long-lasting synaptic PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 Capecitabine plasticity and memory. Neuron 2009, 61:10?6. 3. Jin I, Kandel ER, Hawkins RD: Whereas short-term facilitation is presynaptic, intermediate-term facilitation involves both presynaptic and postsynaptic protein kinases and protein synthesis. Learn Mem 2011, 18:96?02. 4. Gkogkas C.