Deep red fluorescent nissl stain

For immunohistochemistry, sections were rehydrated for 3 × 15 min in PBS-0.5% Triton-X100 or PBS-0.1% Tween-20 (PBS-T) at room temperature (RT) and, then, blocked for 1 h in 1% normal goat serum and 1% normal donkey serum in PBS-T at RT. Sections were incubated with the primary antibody (prepared in blocking solution), overnight at 4°C. The following primary antibodies (1:1,000 µl) were used: rabbit polyclonal anti-RFP (#600-401-379S, Rockland); Living Colors DsRed Polyclonal Antibody (#632496, Takara); chicken anti-GFP (#ab13970, Abcam). Sections were washed for 3 × 15 min with PBS-T at RT and then incubated with the appropriate secondary antibodies (prepared in PBS-T) for 1 h at RT. The used secondary antibodies were: Cy3 AffiniPure Goat Anti-Rabbit IgG (H + L) (1:300; #111-165-003, Jackson ImmunoResearch); Alexa Fluor 488 anti-chicken (1:500; #A11039, Invitrogen); Alexa Fluor 568 anti-rabbit (1:200; #A10042, Invitrogen). Finally, slides were washed 3 × 15 min with PBS-T and either counterstained with NeuroTrace (1:200, 435/455 Blue Fluorescent Nissl Stain (#N21479) or 640/660 Deep-Red Fluorescent Nissl Stain (#N21483), ThermoFisher Scientific) and mounted in Prolong Diamond mounting media (#P3691, ThermoFisher Scientific) or mounted directly in Vectashield Plus Antifade mounting medium with DAPI (#H-2000, Vector Laboratories).

Cultured N2a cells were transfected with pCS2 or pCS2-HAmOXTR. Live cells were then incubated with aCSF containing 1 mM oxytocin (GL Biochem) for 20 min, and then fixed in 4% PFA for 1 h. As control, cells were incubated in aCSF containing 1 mM Vasopressin (GL Biochem). Immunostaining was performed with the following antibodies: oxytocin (Phoenix Pharmaceuticals, Cat# G-051–01, RRID: AB_2876858; 1:250), HA (Covance, Cat# MMS-101P, RRID: AB_2314672; 1:200), Donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Cat# A-21206, RRID:AB_2535792; 1:500) and Donkey anti-mouse Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A10037; 1:500). For the antibody block experiment, the oxytocin antibody was pre-incubated with 10 mM oxytocin. Sections were incubated with 640/660 Deep-Red Fluorescent Nissl Stain (Thermo Fisher Scientific Cat# N21483) for 20 min at room temperature, washed, and mounted onto glass slides in 70% glycerol. Images were acquired on a Nikon A1 confocal microscope with a Plan Apo VC 20× DIC N2 (N.A. = 0.8) objective.