Nickel chloride

For PCNA immunohistochemistry, we chose the sections immediately adjacent to the one identified as having the highest number of GnRH3 neurons. These sections were incubated in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. Next, sections were immunohistochemically stained with mouse monoclonal anti-PCNA antibody (1:3000, P8825, Sigma-Aldrich) diluted in 0.1 M PBS containing 0.03% Triton X-100 and 10% Block Ace and with biotinylated goat anti-mouse IgG (1:500, Vector Laboratories, Burlingame, CA, USA). After incubation with an avidin-biotin-peroxidase-complex (ABC Method kit, Vector Laboratories), the sections were incubated with 0.02% solution of 3, 3′-diaminobenzidine (Wako Pure Chemical Industries) in 0.175 M sodium acetate buffer containing 0.125% nickel chloride (Wako Pure Chemical Industries, Osaka, Japan) and 0.0026% hydrogen peroxide (Wako Pure Chemical Industries).
In every PCNA-stained sections obtained per animal, the number of cells with PCNA-positive nuclei (PCNA-positive cells) were counted in the dorsal and ventral areas (each area, 140 × 140 µm) around the ventricle (Fig. 1a). Average dorsal and ventral densities of PCNA-positive cells per animal were calculated, and mean densities and standard errors of PCNA-positive cells were then determined for control (n = 3) and 11-KT injected (n = 3) animal groups.

Nickel chloride (NiCl₂), zinc chloride (ZnCl₂), cobalt chloride (CoCl₂), copper (II) chloride dihydrate (CuCl₂·2H₂O), iron (II) chloride tetrahydrate (FeCl₂·4H₂O), magnesium chloride hexahydrate (MgCl₂·6H₂O), manganese (II) chloride tetrahydrate (MnCl₂·4H₂O), lipopolysaccharides (LPS) from Escherichia coli O111, and 30% (w/v) H₂O₂ were purchased from Wako Pure Chemical Industries (Osaka, Japan). Chlorazol Black and TAK-242 were purchased from Sigma-Aldrich Co. (St. Louis, MO) and Calbiochem-Merck Millipore (Darmstadt, Germany), respectively. Newport Green^TM DCF diacetate was purchased from Invitrogen (Carlsbad, CA) and the Ni wire (purity 99.98%, diameter 0.8 mm) from Nilako (Tokyo, Japan). HNO₃ (69% (w/w)) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).

Cellulose cotton fiber (75 g/m²), produced by the El-Mahalla Company for Spinning and Weaving—EL-Mahalla ELkobra, Egypt, was used. Acrylonitrile (AN), acrylic acid (AA), and styrene (Sty) were received from Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and were purified by distillation. Potassium permanganate, dimethylformamide (DMF), and ethyl acetate were purchased from Kishida Chemical Co., Ltd., Osaka, Japan and used as received. Citric acid, acetone, methanol, sodium hydroxide, nickel chloride, and copper chloride were purchased from Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and were used without further purification.