Donkey anti rabbit cy5

The desired brain sections containing the SFO and OVLT were initially washed free‐floating in PBS. Next, brain tissue was permeabilized with 0.1% Triton X100 (Sigma) for 20 minutes at room temperature (RT), following further washing in PBS. Slices were then blocked in 5% normal donkey serum (NDS; Sigma) and 0.05% Triton X100 at RT for 30 minutes. A 48 hours incubation at 4°C in a PBS solution containing 0.05% Triton X100, 0.5% NDS and the primary antibodies rabbit anti‐GFP (1:1000, Abcam), and chicken anti‐vimentin (1:4000, Abcam). Following this incubation, slices were washed in PBS and transferred to PBS solution of secondary antibodies for 24 hours at 4°C; donkey anti‐rabbit Cy5 (1:800, Jackson ImmunoResearch) and donkey anti‐chicken Alexa 488 (1:800, Abcam). The secondary antibodies were then washed off in PBS and the slices were mounted on gelatine‐coated glass slides using VectaShield medium (Vector Laboratories, USA). Slices were then imaged on a confocal microscope (Leica SP5 upright) under the 20x objective. Z‐stacks were acquired in 3 µm steps and viewed as maximal intensity projections.

One series of free-floating sections were rinsed in 0.1M PBS then incubated in 0.3% H₂0₂ in PBS for 10 min. After rinsing, tissue was blocked with 2% normal donkey serum, 0.3% Triton-X 100 in PBS, then transferred into primary antibody against GnIH (PAC123/124, Bentley) 1:5000 in PBS plus 0.3% Triton-100 [PBS-T] and sections were incubated in antibody overnight, on rotation, at 4°C. The next day, sections were rinsed in PBS and incubated in secondary for 1 hr at room temperature (Biotin donkey anti-rabbit 1:500, Jackson ImmunoResearch, West Grove, PA). Following rinsing, sections were incubated in ABC reagent (Vector) and then amplified by incubating in biotinylated tyramide for 30 min. Tertiary incubation for 1 hr at room temperature followed with streptavidin-Alexa594 (1:1000 in PBS, Jackson Immunoresearch). Following tertiary incubation, sections were incubated in an antibody against blue-fluorescent protein (anti-BFP; 1:5000, Abcam, Cambridge, MA) on a rotating stage, overnight, at 4°C. The next day, sections were rinsed in PBS then incubated in secondary antibody for 2 hr at room temperature (donkey anti-rabbit cy5, Jackson Immunoresearch). After rinsing in PBS-T, slides were coverslipped using DABCO antifading medium and stored in the dark at 4°C.