Eclipse e800m

Every 6th section of the diencephalon was processed for kisspeptin/NKB double-labeling fluorescence immunohistochemistry using the tyramide signal amplification (TSA) method [8 (link), 33 (link)]. Sections were sequentially incubated with 10% NGS in PBST-BSA for 1 h, anti-NKB polyclonal antibody (1:6,000) in PBST-BSA containing 2% NGS at 4°C for 72 h, biotinylated goat anti-rabbit IgG
(1:200 in PBST-BSA containing 2% NGS) for 3 h, and avidin-biotin complex (2 µl/ml PBST) for 1 h. After being soaked in the TSA blocking solution for 30 min, the sections were reacted with biotinyl tyramide solution (1:200,
PerkinElmer, Waltham, MA, USA) for 10 min, and streptavidin-Alexa 488 (1:200, Invitrogen, Carlsbad, CA, USA) in PBST for 1 h. They were then incubated with gC2 (1:2,000) for 48 h at 4°C and Alexa-555 conjugated anti-rabbit IgG
(1:200, Invitrogen) for 3 h. Kisspeptin/Dyn double-labeling immunohistochemistry was conducted using the same protocol and the anti-Dyn polyclonal antibody (1:20,000). Sections were mounted on gelatin-coated slides and
cover-slipped with a water-soluble mounting medium (Vector Laboratories). The sections were observed under a microscope (ECLIPSE E800M, Nikon, Tokyo, Japan) equipped with a charge-coupled device camera (AxioCam HRc, Carl Zeiss,
Jena, Germany). The two fluorescent images were merged using computer software (AxioVision, Carl Zeiss).

Tissues from two goats were used in an experiment to make observations of the structure.
The sections were stained with methyl green-pyronin Y (Muto Pure Chemicals, Tokyo, Japan)
for 15 min at room temperature and then washed with water and cover slipped using Entellan
new (Merck, Darmstadt, Germany) as the mounting medium. The sections were observed by
taking photographs using a microscope (ECLIPSE E800M; Nikon, Tokyo, Japan) equipped with a
charge-coupled device camera (AxioCam HRC; Zeiss, Jena, Germany).