The main objective of our study was to evaluate the C57BL/6 http://www.selleckchem.com/products/Sorafenib-Tosylate.html mice as a model of dengue virus infection.2. Material and Method2.1. Mice Three-to-four-week-old C57BL/6 mice were obtained from the Central Animal Facility at the University of Sao Paulo/Ribeirao Preto and housed in specific pathogen free (SPF) conditions at the Research Center of Virology, Medical School of Ribeirao Preto (FMRP). All the procedures were approved by the Animal Ethics Committee of the FMRP (Protocol: 103/2005).2.2. Virus DENV-1, Mochizuki strain, was used in this study. The virus was maintained in C6/36 cells culture at 28��C for 7 days. Then, the virus was propagated in newborn (1 to 2 days old) Swiss mice, by intracerebral inoculation of infected cell culture supernatant.

After the first appearance of signs of paralysis, mice were sacrificed and stored at ?70��C until use. The brains of infected and uninfected animals were removed with sterile syringes and prepared as a suspension of 20%, using PBS containing 4% bovine fetal serum. The brain was then macerated and the suspension was centrifuged at 10,000��g for five minutes. The supernatant was aliquoted and stored at ?70��C. The viral title was determined by the plaque assay [6].2.3. Animal Infection and Sample Processing The animals were infected intraperitoneally with 150��L viral suspension containing 7.2 �� 107PFU or 150��L of uninfected brains suspension. At different times postinfection, the blood was obtained from the retroorbital region and collected in a tube containing sodium citrate (3.

8%) as an anticoagulant in an amount corresponding to 10% of the Batimastat total volume; then, the animals were sacrificed to remove liver, brain, spleen and kidney. The blood was centrifuged at 1000��g for five minutes to obtain the plasma. The whole organs were suspended in 500mL of PBS and ground in a tissue homogenizer, except liver that only the right lobe was used. The suspension was centrifuged at 8000��g for five minutes and the supernatant was stored at ?70��C until use for viral load quantification. Liver and brain of four mice were fixed in 4% neutral formalin solution, embedded in paraffin, and sectioned at a thickness of 3��m. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin (H&E). The sections were then dehydrated before mounting. The total number of nucleated cells was counted in fifty microscopic fields in at least four representative, nonconsecutive, HE-stained sections from each mouse. Sections were examined using a Zeiss Integrationsplatte II eyepiece (Zeiss Co, Oberkochen, Germany) reticule, using a microscope at a final magnification of 400x.