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2 protocols using sc 134499

1

Immunofluorescent Staining of Hypothalamic Sections

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Serial hypothalamic sections (10 sections per sample) were blocked with 10% donkey serum and then incubated with antibodies against GnRH (dilution 1 : 100, sc-32292, Santa Cruz Biotechnology Inc., Dallas, TX, USA), Kiss-1 (dilution 1 : 100, sc-18134, Santa Cruz Biotechnology Inc.), and GPR54 (dilution 1 : 100, sc-134499, Santa Cruz Biotechnology Inc.) for 24 h at 4°C. The sections were then treated with the following secondary antibodies: Alexa 488-conjugated donkey anti-mouse IgG (A-21202, Thermo Fisher Scientific Inc., Waltham, MA, USA), Alexa 633-conjugated donkey anti-goat IgG (A-21082, Thermo Fisher Scientific Inc.), and Alexa 594-conjugated donkey anti-rabbit IgG (A-21207, Thermo Fisher Scientific Inc.). After being stained with DAPI (D1306, Thermo Fisher Scientific Inc.), the sections were imaged and analyzed with a laser scanning confocal microscope (SP8, Leica Microsystems Inc., Buffalo Grove, IL, USA).
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2

Investigating Kisspeptin Signaling Pathway

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Kp-10 (Metastin 45-54 (H-YNWNSFGLRF-NH 2 )) of human origin, a Kp form with a potency similar to Xenopus-Kp-10 for bullfrog Gpr54 (Moon et al. 2009) (link), was purchased from DBA Italia, Milan, Italy; Kp-234, a specific Gpr54 antagonist (Roseweir et al. 2009 (link)) was purchased from DBA Italia; 17B-estradiol (E 2 ) was purchased from Sigma-Aldrich S.R.L.; ICI 182780, a high-affinity estrogen receptor antagonist, was purchased from AstraZeneca, UK; and buserelin, a GnRH agonist, was a gift from Dr J Sandow, Hoechst, Frankfurt, Germany.
For Western blot and immunohistochemistry the following antibodies were used: goat polyclonal anti-Kiss-1 antibody (raised against a peptide mapping at the C-terminus of Kiss-1 of human origin; sc-18134) and rabbit polyclonal anti-Gpr54 antibody (raised against amino acids 141-342 mapping within an internal region of Gpr54 of human origin sharing 78% similarity with P. esculentus Gpr54; sc-134499); both were purchased from Santa Cruz Biotechnology and were validated for Western blot and immunohistochemistry (present data, Supplementary Figs 1 and2, see section on supplementary data given at the end of this article; Hsu et al. 2014 (link), Ciaramella et al. 2016) . Mouse monoclonal anti-α-tubulin (T6199) was purchased from Sigma-Aldrich.
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