Samples from all experimental groups were method processed in parallel to minimize inter assay variation. The results represent the mean of three to five inde pendent experiments. Tissue collection Deeply anesthetized animals were transcardially perfused with ice cold saline, 54 h after pMCAO induction, fol lowed by 4% paraformaldehyde in 0. 1 M phosphate buffer. The rats brains were removed and post fixed overnight at 4 C. The following day, the brains were washed three times in PBS, and cryo protected in 30% sucrose in PBS at 4 C for 48 72 h. Finally, the brains were embedded in Tissue Tek medium and stored at ?20 C. Coronal cryostat sections were obtained from each brain and three tissue sections were collected onto each Superfrost Ultra Plus slides, air dried and stored at ?20 C.
Sections located from 2 to ?2 relative to bregma were selected for immunochemistry. Immunohistochemistry Immunohistochemical Inhibitors,Modulators,Libraries analyses were performed in Inhibitors,Modulators,Libraries a hu midified chamber. The sections were dried at room temperature for at least 1 h, washed once in PBS, permeabilized for 15 min in 0. 25% triton X 100 in PBS and incubated for 30 min in blocking solution containing 0. 1% Triton X 100 in PBS supplemented with 1% horse serum. The slides were incubated over night at 4 C with the appropriate primary antibody diluted in PBS containing 0. 1% Triton X 100 and 1% horse serum, monoclonal mouse anti GFAP, anti Iba1 rabbit, and a monoclonal rabbit anti phospho AktSer473. The sections were then washed three times in PBS containing 0.
1% Triton X 100, and again in PBS, and endogenous peroxidase activity was quenched by incubating for 30 min in PBS containing 1% Inhibitors,Modulators,Libraries hydrogen peroxide. The slides were subsequently incubated for 1 h at room temperature with the appropriate secondary antibody, biotin conjugated anti mouse IgG or biotin con jugated anti rabbit IgG. After washing five times with PBS, the sections were incubated for 30 min with avidin biotin peroxidase complex from the Vector ABC Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries Elite kit. The sections were then washed in PBS and incubated for 15 min with diaminobenzidine reagent FAST 33 diaminobenzidine tablets. Fol lowing dehydration in a series of graded ethanol dilu tions, the sections were cleared with xylol and mounted using Entellan New mounting medium. For double immunostaining experiments, follow ing incubation with the primary antibody, the sections were incubated for 1 h with anti rabbit Alexa 488 and anti mouse Alexa 555 fluorescent secondary antibodies.
After washing three times with sellckchem PBS, the slices were mounted using Fluoromount G. To determine the specificity of the immunoreac tions, each batch experiment included control prepara tions in which the primary antibody was omitted and the incubation solution replaced with blocking solution. Slides immunostained with Iba1 or GFAP were observed under an Olympus BX61 microscope and images captured using an Olympus DP50 camera.