, 2003). The i.p. route was used given the difficulty for injecting the venom through the small-sized tail vein of the 14 days old neonate rats. Animals of both ages (P14 and 8–10 wks old received a single i.p. injection of PNV (1.7 mg/kg in 0.5 ml saline solution (vehicle) or 0.5 ml of 0.9% sterile saline (sham group); one, two, five and 24 h after injection (n = 5 per time/treatment), the buy Obeticholic Acid animals were anesthetized with i.p. injection (2 μg/mg
body weight) of a 3:1 mixture of ketamine (Dopalen, 100 mg kg−1 body weight) and xylazine hydrochloride (Anasedan, 10 mg kg−1 body weight) (Fortvale, Valinhos, SP, Brazil). This study was approved by the institution’s Committee for Ethics in Animal Use (CEUA/Unicamp, protocol no. 2403-1) and the experiments were done according to the Brazilian Society for Laboratory Animal Science guidelines (SBCAL; formerly Brazilian College for Animal Experimentation – COBEA). The envenoming signs presented by each animal were independently monitored by three observers (M.C.P.M., E.S.S., L.M.S.) and a consensual final register was emitted. Anesthetized animals were transcardially perfused with physiological saline followed by 4% paraformaldehyde
in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Then, the brains were immediately removed and post-fixed in the same fixative overnight at 4 °C. After, they were washed, dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin (Paraplast®, Sigma Aldrich, St. Louis, MO, USA). Selected coronal sections (5 μm) from hippocampus containing the regions (CA1, Adriamycin clinical trial CA2, CA3 and dentate gyrus (DG)) were obtained with the help of a stereotaxic atlas of Afatinib rat brain anatomy (Paxinos and Watson, 1998). Coronal sections of the hippocampus from all groups mounted onto subbed glass slides were dewaxed with xylene and rehydrated in descendent ethanol series until distilled water. One section from each sectioning plane per animal was stained by hematoxylin and eosin (H&E) for histological analysis. For immunohistochemistry, the endogenous peroxidase was blocked with 3% hydrogen peroxide, (two cycles of
10 min) and epitope retrieval was accomplished with 10 mM sodium citrate buffer, pH 6.0, in a steamer (95–99 °C) for 30 min. Non-specific antigen binding was blocked with 5% reconstituted milk powder for 1 h. Slides were incubated with the Flt-1 primary antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 16–18 h in a humidified chamber at 4 °C. After returning to room temperature (RT), slides were washed before being incubated with biotinylated anti-rabbit secondary antibody (EnVision™ HRP link, Dako Cytomation, CA, USA) for 30 min at RT. Color was developed with a diaminobenzidine chromogenic solution (DAB+, Dako Cytomation, CA, USA) and nuclei were counterstained with Harry’s hematoxylin; after ethanol dehydration slides were mounted in Canada balsam.