Goat anti orexin

For double labelling of orexin and tyrosine hydroxylase (TH), tissues were washed and incubated with a PBS cocktail consisting of 2% NDS, 0.3% Triton X-100, goat anti-orexin (1:100 of Cat #Sc-8070 Santa Cruz Biotechnology Inc.) and mouse anti-TH (TH: 1:1000; Cat #T1299, Sigma-Aldrich) antibodies at 4°C for 48 h. The sections were then washed and incubated in Alexa Fluor 594 donkey anti-goat secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) in 0.1M PBS for 2½ h. After washing in PBS, sections were incubated with Alexa-Fluor 488 donkey anti-mouse secondary antibody (1:100, Jackson) for 2½ h. Finally, the sections were rinsed in PBS and cover-slipped using Vecta Shield (Vector Laboratories) anti-fade mounting media.
Controls for each experiment were performed to determine whether the primary or the secondary antibodies produced false-positive results. The controls involved omission of the primary and/or secondary antisera to eliminate the corresponding specific labelling. Nonspecific activation of c-Fos was assessed by evaluating the CNS expression of c-Fos in animals receiving IP injection of PS.

Following standard perfusions as described in [18 (link)], 0.1 mm sections were cut on a vibratome. Sections were blocked in PBS with 0.3% Triton X-100 and 1% bovine serum albumin (blocking solution) for 1 h, incubated with goat anti-orexin (1:1000; Cat# 8072, RRID: AB_653601, Santa Cruz) over-night, washed, incubated with Alexa 647 conjugated donkey anti-goat (1:1000; Cat# A-21447, RRID: AB_2535864, Invitrogen/Thermo Fisher Scientific) for 3.5 h, washed and mounted. Antibodies were applied in blocking solution. Confocal micrographs were acquired on a Nikon A1 and merged in imageJ.