7% formaldehyde and quickly frozen. Tissues were sectioned coronally at 20 µm thickness and sequentially
dipped into xylene 5 min, 100% alcohol 5 min, 95% alcohol 5 min, and 70% alcohol 5 min. Samples were stained with cresyl violet (Sigma-Aldrich, St. Louis, MO, USA) for 3 min. After the staining, slides were reacted with 70% alcohol 5 min, 95% alcohol 5 min, 100% alcohol 5 min, and xylene 5 min. After these processes, sections were observed under a microscope equipped with a digital camera (Olympus, Tokyo, Japan). Five-micrometer-thick frozen brain sections were cut onto clean glass slides (Thermo Scientific, Waltham, MA, USA), air-dried, and fixed in cold acetone for 10 min at –20 °C. The slides were first washed in Tris-buffered saline (TBS) and then incubated with 0.3% H2O2 Bortezomib in methanol to quench endogenous peroxidase activity. Followed by a series of washes (three times with distilled water), the sections were blocked with 10% normal rabbit serum. Frozen brain sections (20 μm) were fixed in ice-cold acetone for 20 min. To block nonspecific labeling, sections were incubated in 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS for 30 min before addition of primary and secondary antibodies.
SB203580 concentration Primary antibodies for VEGF (1:50, Millipore, Billerica, MA, USA), AQP-1 (1:50, Abcam, Cambridge, MA, USA) were applied to the samples for 24 h at 4 °C, Arachidonate 15-lipoxygenase followed by a 90 min incubation with appropriate florescence secondary antibody (1:100, Invitrogen, Carlsbad, CA, USA) and three washes in PBS for 10 min each. After three washes in 0.1% PBS with Tween-20 (PBST), the sections were incubated with rhodamine-conjugated sheep anti-rabbit or FITC-conjugated sheep anti-mouse secondary antibody that was diluted to 1:200 with 5%
BSA fraction V in 0.1% PBST for 2 h in the dark at RT. After three washes in PBS, all sections were incubated with 1 μg/mL of 4׳,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA) and 2 μg/mL of propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) for a counter staining. Tissues were then visualized under a confocal microscope (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany). Statistical analyses were carried out using SPSS 18.0 software (IBM Corp., Armonk, NY, USA). All data are expressed as mean±S.E.M. Significant intergroup differences were determined by one-way analysis of variance followed by Bonferroni post hoc multiple comparison test. Statistical significance with the OGD/R or MCAO group was determined by t-test. Each experiment included at least three replicates per condition. Differences were considered significant at ⁎p<0.05, ⁎⁎p<0.01. The authors declare no conflict of interest regarding the publication of this paper.