Ry simply because food deprivation was utilized in our behavioral tasks as a mild stressor [22]. Physique weight was measured and recorded on the final day of your week. The rats had been sacrificed after the behavioral tests. The whole brains of three rats in each group have been removed following accepting heart perfusion. The brains had been fixed in 4 paraformaldehyde resolution for additional histopathological assessment and immunohistochemistry. The left hippoacmpi of the remaining rats had been flash-frozen in liquid nitrogen and stored at -80 C for protein evaluation. The appropriate hippocampi have been kept in ten volumes of RNAlater Answer (Ambion, Life technologies, Carlsbad, CA, USA) for polymerase chain reaction (PCR) evaluation. two.four. Hormone Measurement. Blood was collected in the abdominal aorta by a puncturing needle. Cerebrospinal fluid (CSF) was extracted from the foramen magnum by a 1 mL syringe. Blood samples were placed at area temperature for 30 min ahead of centrifugation for ten min at three,000 . Serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were analyzed using an IMMULITE 2000 immunoassay technique with ACTH and CORT immunoassay kits. The serum as well as the CSF levels of CRH and urocortin-2 (UCN2) have been detected by a microtiter plate reader (Victor3V , Perkin Elmer, Waltham, MA, USA) with rat CRH enzymelinked immunosorbent assay (ELISA) kit (Baoman Biotech, Shanghai) and rat UCN2 ELISA kit (Chang Yi Chemical, Chemical, Shanghai). two.five. Histopathological Assessment and Nissl’s Staining. Inside the histopathological examination, paraformaldehyde answer was employed to repair the paraffin-embedded brain tissues that were cut into serial sections (3 m). The slices had been stained with hematoxylin and eosin and Nissl’s resolution within a routine process. 2.6. Two-Dimensional Electrophoresis (2D) and Protein Identification. The 2D electrophoresis was performed as previously reported and repeated for three instances [18]. Briefly, hippocampal samples containing 300 g of protein had been loaded per tube in an isoelectric focusing program (IPGphor II, GE). The samples were isoelectrofocused and separated with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The gels were silver-stained. The protein spots that either elevated or decreased for far more than twofold had been selected for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) identification. The3 mascot software program package plus the database of SwissProt have been employed to match the mass of peptides [23]. two.7. Western Blot Evaluation. Western blot analysis was performed as previously described [24]. Briefly, the protein lysates have been loaded onto 10 SDS-PAGE for separation, electrotransferred onto PVDF membranes, and blocked in five nonfat milk in Tris-buffered saline Tween. The membranes were incubated overnight with principal antibodies, like anti–arrestin 2 (Cell Signaling Technology, CST, clone C16D9, 1 : 750 dilution), anti-extracellular signal-regulated kinase (ERK, CST, Clone 137F5, 1 : 800 dilution), antiphospho-ERK (Thr202/Tyr204, CST, 1 : 800 dilution), antityrosine receptor kinase B (TrkB, Bioss, clone bs-0175R, 1 : 400 dilution), anti-BDNF (epitomics, 1 : 4000 dilution), anti-serine/threonine-protein 4-Hydroperoxy cyclophosphamide MedChemExpress phosphatase 2A subunit B (PP2A b, Abcam, 1 : 800 dilution), anti-PP2A c (Abcam, 1 : 750 dilution), anti-CRH receptor 1 (CRHR1, Bioss, 1 : 400 dilution), anti-CRH receptor two (CRHR2, Abcam, 1 : 800 dilution), anti-mammalian target of rapamycin (mTOR, CST, 1 : 800 dilution), anti-phospho-mTOR (phospho-S244.
