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Immunological staining was performed with OPG and RANKL polyclonal antibodies (Santa Cruz, USA), the streptavidin-peroxidase (S-P) immunohistochemical reagent kit, and the diaminobenzidine (DAB) developing box (Beijing Zhongshan Biochemicals Co.
The second antibody was biotinylated goat antirabic serum (Vector, Burlingame, CA, USA) (1:250); visualisation was with nickel-enhanced diaminobenzidine (Ni-DAB) for Fos, and diaminobenzidine (DAB) for double-labelling of kisspeptin.
Following washing, the strips were incubated with diaminobenzidine substrate for 5-10 min.
We visualized the presence of antigen on the cell surfaces using a Vectastain ABC kit (Vector Laboratories) with diaminobenzidine tetrahydrochloride (DAB) as a substrate followed by counterstaining with hematoxylin.
Dulbecco's Modified Eagle Medium (DMEM), fetal calf serum (FCS), glutamine, [iota]-glutamic acid (monosodium salt), poly-D-lysine, diaminobenzidine, propidium iodide (PI), pyruvic acid ([alpha]-ketopropionic acid) sodium salt, [beta]-nicotinamide adenine dinucleotide (reduced form), paraformaldehyde, 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl, free radical (DPPH) and ([+ or -])-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma (USA).
05% Tween-PBS, and blots were developed by diaminobenzidine substrate solution containing [H.
The secondary antibody was detected using a Vectastain kit in combination with diaminobenzidine (Vector Labs, Burlingame, CA).