The time for the rat to flee onto the submerged platform was noted with a computer program connected to a camera mounted in the roof directly above the pool, as described previously. Immunohistochemistry One or 24 hours after hypoxia, brains were Tipifarnib molecular weight taken after the subjects have been perfused with 4% paraformaldehyde, and post fixed overnight at 4 C, followed by incubation with thirty days sucrose phosphate buffer for 48 hours. Serial frozen sections were obtained on gelatin coated slides. BBB permeability measured by IgG extravasation staining was performed 24-hours post hypoxia. Brain sections were incubated with 0. One month H2O2/methanol for half an hour, and then anti IgG antibody for 2 hours. Biotin peroxidase signals were found using 0. 5 mg/ mL 33 diaminobenzidine/0.. 003% H2O2 as a substrate.. Measurements of the integral optical density of IgG signals in the cortex were analyzed using imaging software at 200 magnification per visual Neuroblastoma field. . The mean IOD was counted and averaged from three visual fields per part, and three brain sections, which corresponded to plates 18, 31 and 39 in a rat brain atlas, of each brain of each experimental group were compared to those of the get a handle on group and expressed as relative IOD proportions. Immunofluorescence staining Immunofluorescence was done on frozen sections. Activated microglia and pJNK at 1-hour post hypoxia, and apoptosis were tested at 24 hours post hypoxia. Brain sections were blocked with two weeks normal goat serum and 0.. One of the Triton X 100, and probed with primary antibodies p JNK, cleaved caspase 3, NeuN, RECA1, GFAP, Iba1, or ED1 in PBS/ 0. 03-18 Triton X 100 at 4 order Dabrafenib C over night. The pieces were then incubated with Alexa Fluor 488 goat IgG and Alexa Fluor 594 goat IgG secondary antibodies for 1-hour at room temperature. Images were acquired on a Nikon E400 fluorescence microscope. Digitally captured images were analyzed using NIS Elements imaging software. ED1 microglia were measured at 200 magnification per visual field in the cortex, and three visual fields per part, and three brain sections, which corresponded to plates 18, 31 and 39 in a ratbrain atlas, of each brain were counted and expressed as an average number per visual field. After the subjects had been perfused with 2% paraformaldehyde and 2% glutaraldehyde in 0 digital microscopy examination Twenty four hours after hypoxia, brains were taken. 1 M pH 7. 2 phosphate buffer, and postfixed in the same fixative for 2 hours. The samples were blocked and fixed in one of the osmium tetroxide aqueous solution for 1-hour, and washed with ddH2O for 10 min three times, then dehydrated in real propylene oxide and increasingly graded ethanol. The samples were embedded in Epon at room temperature and polymerized within an oven at 55 C for 1 day. Eighty nm thick sections were cut and collected onto the grids. The parts were then stained with lead citrate and uranyl acetate and noticed with a JOEL 1200 EX transmission electron microscope.