Investigating Neuropeptide Y Neuron Co-Expression

To investigate NPY^ARN co-expression with gonadal steroid hormone receptors, free-floating immunohistochemistry was performed on coronal sections throughout the rostral, middle, and caudal ARN (rARN, mARN, cARN). NPY^ARN neuron somata were labeled for GFP reporter expression using a chicken anti-GFP primary antibody (1:5000; Aves Labs) [45 (link)] as above. Every third section was co-labeled for one of the following steroid hormone receptors: progesterone receptor (rabbit anti-PR, 1:100; Dako (Agilent), CA, USA; VEH n = 8, PNA n = 10; RRID:AB_2315192) [46 (link)], estrogen receptor α (rabbit anti-ERα, 1:5000; Millipore; VEH n = 5, PNA n = 5, RRID:AB_310305) [47 (link)], or androgen receptor (rabbit anti-AR PG-21, 1:200; Millipore, MA, USA; VEH n = 4, PNA n = 4; RRID:AB_310214) [48 (link)]. To amplify GFP signal, a goat anti-chicken AlexaFluor488 antibody was used (1:200; Molecular Probes, OR, USA), while steroid hormone receptors were labeled using a goat anti-rabbit AlexaFluor568 antibody (1:200; Molecular Probes, OR, USA).