Confirmation of L10a-GFP expression in GnRH neurons

GnRH-cre/L10a-GFP mice (n = 3/treatment) were perfused transcardially with 4% paraformaldehyde and brains removed. Brains were cryopreserved overnight in 20% sucrose, then frozen in optimum cutting temperature (Tissue-Tek; Sakura Finetek, Torrance, CA), and sectioned at 30 µm into four series on a cryostat (Leica, Buffalo Grove, IL). One series from just caudal to the olfactory bulb through the optic chiasm was processed for free-floating dual immunofluorescence using standard procedures (37). The primary antibodies (Table 1) used were the following: chicken anti-GFP (ab13970, 1:1000; Abcam, Cambridge, UK) and rabbit anti-GnRH (EL-14, 1:4000; generous gift from Dr. Oline Ronnekleiv, Oregon Health & Science University, Portland, OR) (38). Primary antibodies were visualized with Alexa Fluor-488 conjugated goat anti-chicken and Alexa Fluor 546-conjugated goat anti-rabbit (A-11039 and A-11035, respectively; Thermo Fisher Scientific, Waltham, MA), respectively. Sections were mounted on Superfrost Plus glass slides (Thermo Fisher Scientific) and coverslipped with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific). Immunofluorescence was detected using a fluorescent Axio Imager microscope (Zeiss, Jena, Germany). The number of cells expressing GnRH, GFP, or both was counted in the three sections surrounding the organum vasculosum of the lamina terminalis containing the highest number of GnRH neurons. The region contained within the tissue punch used for TRAPseq was focused on, defined as ventral to the anterior commissure, and extended 0.6 mm lateral from midline on each side.

Antibody Table

Abcam, Cambridge, UK; Oregon Health & Science University, Portland, OR; Antibody and Bioresource Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY.

Abbreviation: RRID, research resource identifier.