Ine serum as standard [18], each sample was diluted to equal protein concentrations with HB. After adding 46sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer into the sample, the sample was boiled in 100uC water for 10 min. Protein (50 mg) was loaded onto each lane, 1326631 separated by 15 548-04-9 web SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, UK). The membrane was blocked with 5 skimmed milk for 2 h, and then probed with rabbit poloclonal anti-BDNF antibody (1:500, ab72439, ABcam,USA ) or mouse monoclonal a-tubulin (1:1000 dilution, sc-23948, Santa cruz,USA) at 4uC overnight. Detection was performed using horseradish peroxidase(HRP) conjugated goat anti-mouse IgG (1:2000 dilution, P0260, Dako, A/S, Denmark) or HRP conjugated goat antirabbit IgG (1:2000 dilution, P0048, Dako, A/S, Denmark), and visualized by an ECL method using ECL Western BlottingTreatments and Tissue PreparationAn overdose of BDNF(1 mg/mouse)was used according to the report that intraperitoneal injection of 100 ng/rat recombinant BDNF can effectively induce a decrease in colonic reaction threshold [16]. From the 21st day, the mice in the BDNF-treated and BDNF-treated stressed groups were treated daily by intraperitoneal injection with 1 mg recombinant BDNF (GenWay Biotech, Inc., USA). The treatment was continued until the day when mice were 1379592 killed. The mice in other groups were injected with vehicle (0.9 NaCl). After the open field test on the 30th day, mice in all groups AKT inhibitor 2 site received 5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally, followed with 10 IU human chorionic gonadotropin (hCG) 48 hours later. The mice used for evaluation of BDNF expression were killed 6 hours after hCG injection. Animals were decapitated and trunk blood was collected, and plasma was stored at 280uC until the time of corticosterone assay. Left ovaries for western blotting were dipped into liquid nitrogen and stored at 280uC. Right ovariesStress on Ovarian BDNF and Oocytes DevelopmentSubstrate (Promega). The bands on the X-ray film were scanned. BDNF bands were normalized relative to a-tubulin.ImmunohistochemistyFor immunohistochemical detection of corticotropin-releasing hormone (CRH), brain sections were incubated in 0.3 H2O2 solution and blocked with 10 normal goat serum in 0.1 Triton X-100. Then the sections were incubated overnight with rabbit poloclonal anti-CRH antibody (1:1000, T-4037, Bachem Inc., Bubendorf, Switzerland) at 4uC. After washing, sections were incubated for 2 h with HRP conjugated horse anti-rabbit IgG (1:2000 dilution, P0048, Dako, A/S, Denmark) at room temperature, visualized with DAB/(NH4)2Ni(SO4)2, dehydrated in ethanol, and mounted in Entellan. For immunohistochemical detection of BDNF, the sections were treated with microwaves (700 W) in 0.05 M citrate-buffered saline (pH 6.0) for 2 610 min for antigen retrieval. After incubating in 0.3 H2O2 solution and blocking with 10 normal goat serum in 0.1 Triton X-100, sections were incubated overnight with rabbit poloclonal anti-BDNF antibody (1:100, ab72439, ABcam,USA ) at 4uC. After washing, sections were incubated for 2 h with HRP conjugated horse anti-rabbit IgG (1:2000 dilution, P0048, Dako, A/S, Denmark) at room temperature, visualized with DAB, dehydrated in ethanol, and mounted in Entellan.follicles. The follicles were classified into four stages according to the modified Oktay system [9]: `primordial follicle’ = an oocyte that was enca.Ine serum as standard [18], each sample was diluted to equal protein concentrations with HB. After adding 46sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer into the sample, the sample was boiled in 100uC water for 10 min. Protein (50 mg) was loaded onto each lane, 1326631 separated by 15 SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, UK). The membrane was blocked with 5 skimmed milk for 2 h, and then probed with rabbit poloclonal anti-BDNF antibody (1:500, ab72439, ABcam,USA ) or mouse monoclonal a-tubulin (1:1000 dilution, sc-23948, Santa cruz,USA) at 4uC overnight. Detection was performed using horseradish peroxidase(HRP) conjugated goat anti-mouse IgG (1:2000 dilution, P0260, Dako, A/S, Denmark) or HRP conjugated goat antirabbit IgG (1:2000 dilution, P0048, Dako, A/S, Denmark), and visualized by an ECL method using ECL Western BlottingTreatments and Tissue PreparationAn overdose of BDNF(1 mg/mouse)was used according to the report that intraperitoneal injection of 100 ng/rat recombinant BDNF can effectively induce a decrease in colonic reaction threshold [16]. From the 21st day, the mice in the BDNF-treated and BDNF-treated stressed groups were treated daily by intraperitoneal injection with 1 mg recombinant BDNF (GenWay Biotech, Inc., USA). The treatment was continued until the day when mice were 1379592 killed. The mice in other groups were injected with vehicle (0.9 NaCl). After the open field test on the 30th day, mice in all groups received 5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally, followed with 10 IU human chorionic gonadotropin (hCG) 48 hours later. The mice used for evaluation of BDNF expression were killed 6 hours after hCG injection. Animals were decapitated and trunk blood was collected, and plasma was stored at 280uC until the time of corticosterone assay. Left ovaries for western blotting were dipped into liquid nitrogen and stored at 280uC. Right ovariesStress on Ovarian BDNF and Oocytes DevelopmentSubstrate (Promega). The bands on the X-ray film were scanned. BDNF bands were normalized relative to a-tubulin.ImmunohistochemistyFor immunohistochemical detection of corticotropin-releasing hormone (CRH), brain sections were incubated in 0.3 H2O2 solution and blocked with 10 normal goat serum in 0.1 Triton X-100. Then the sections were incubated overnight with rabbit poloclonal anti-CRH antibody (1:1000, T-4037, Bachem Inc., Bubendorf, Switzerland) at 4uC. After washing, sections were incubated for 2 h with HRP conjugated horse anti-rabbit IgG (1:2000 dilution, P0048, Dako, A/S, Denmark) at room temperature, visualized with DAB/(NH4)2Ni(SO4)2, dehydrated in ethanol, and mounted in Entellan. For immunohistochemical detection of BDNF, the sections were treated with microwaves (700 W) in 0.05 M citrate-buffered saline (pH 6.0) for 2 610 min for antigen retrieval. After incubating in 0.3 H2O2 solution and blocking with 10 normal goat serum in 0.1 Triton X-100, sections were incubated overnight with rabbit poloclonal anti-BDNF antibody (1:100, ab72439, ABcam,USA ) at 4uC. After washing, sections were incubated for 2 h with HRP conjugated horse anti-rabbit IgG (1:2000 dilution, P0048, Dako, A/S, Denmark) at room temperature, visualized with DAB, dehydrated in ethanol, and mounted in Entellan.follicles. The follicles were classified into four stages according to the modified Oktay system [9]: `primordial follicle’ = an oocyte that was enca.