Pa3 214

Ovaries obtained after controlled ovarian stimulation were sectioned for immunostaining. Immunostaining was performed on paraffin-embedded tissues sectioned at 5 μm as previously described.^{18 (link)} Immunodetection of NTS and NTSR2 were performed after acidic antigen retrieval (10 mM sodium citrate, 0.05% Tween20; 18), and SORT immunodetection was performed after basic antigen retrieval (10 mM Tris base, 1 mM EDTA, 0.05% Tween20; 18). Slides were blocked, then incubated overnight with rabbit primary antibodies against NTS (ImmunoStar #20072; 1:100 dilution), NTSR1 (Thermo Fisher #PA3–214; 1:400 dilution), NTSR2 (EMD Millipore #AB15134; 20 μg/mL), or SORT1 (Sigma #HPA006889; 4 μg/mL). Omission of the primary antibody served as a negative control. Slides were incubated with DAB substrate using the Vectastain Rabbit ABC kit (Vector Laboratories, Burlingame, CA) according to manufacturer instructions, then counterstained in hematoxylin.

All testis tissues were formalin fixed, paraffin embedded, and sectioned at 5 μm. Immunocytochemical detection of NTS receptors was performed essentially as previously described (Bender et al., 2018). Briefly, tissue sections were heated and deparaffanized. Immunodetection of NTSR1 proceeded without antigen retrieval, NTSR2 was performed after acidic antigen retrieval (Bender et al., 2018), and SORT immunodetection was performed after basic antigen retrieval (Bender et al., 2018). Slides were blocked with 5% non-immune serum in PBS containing 0.1% Triton X-100, then incubated overnight with primary antibodies against NTSR1 (Thermofisher #PA3-214; 1:400 dilution), NTSR2 (EMD Millipore #AB15134; 20 μg/ml), or SORT1 (Sigma Aldrich #HPA006889; 4 μg/ml). Omission of the primary antibody served as a negative control. Slides were incubated with DAB substrate using the Vectastain Rabbit ABC kit (Vector Laboratories, Burlingame, CA) according to manufacturer instructions. Slides were counterstained in hematoxylin, dehydrated, and permanently sealed with a coverslip. All images were obtained using an Olympus BX41 microscope fitted with a DP70 digital camera and associated software.