Guinea pig anti vglut1

Sections were incubated with 0.3% Triton X-100 in PBS for 30 min, blocked with 2% normal goat serum in PBS for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies: rabbit anti-c-fos (Abcam, 1:200) and guinea pig anti-vGlut1 (Millipore, 1:100). Sections were then incubated for 2 h at room temperature with the corresponding secondary antibodies: donkey anti-rabbit IgG conjugated with Alexa Fluor 594 (Invitrogen, 1:500) and goat anti-guinea pig IgG conjugated with tetraethyl rhodamine isothiocyanate (TRITC) (ImmunoReagents, 1:300). For negative controls, adjacent sections were treated with the same steps except by replacing the primary antibodies with PBS. Two symmetric images (×200) in the vlPAG area were acquired at the same exposure in each sight of 5–7 sections per subgroup using Nikon Eclipse 80i fluorescence microscope (Nikon, Japan).

The mouse Crygn peptide CDNFQDQGFMNRVN that was predicted to be antigenic and to discriminate among related proteins was synthesized at Pineda (Berlin, Germany), conjugated to carrier, and used to immunize rabbits. The rabbit anti-Crygd/e was produced by Genescript (Piscataway Township, NJ, USA) by immunization with the peptide RGFQGRHYECSTDHC that is present in both Crygd and Cryg/e. Antibodies were cleaned up by affinity purification. The primary antibody rabbit anti-Cryg was kindly provided by Dr. Samuel Zigler. All three primary antibodies were used in the following dilutions: immunohistochemistry: 1:50 for anti-Crygd/e and Crygn; 1:200 for anti-Cryg, immunocytochemistry: 1:100, and immunoblotting 1:250 for all three antibodies. The primary antibodies guinea pig anti-GlyT2 (Millipore; Darmstadt, Germany) (1:1,000), guinea pig anti-VGluT1 (Millipore) (1:5,000) and the rabbit anti-GlyT2 (1:1,000) have been characterized previously [59 (link),60 (link)]. Mouse anti-flag was obtained from Genescript (1:500).

Adult male Wistar rats were anaesthetised using sodium pentobarbitone (60 mg/kg IP) and perfused transcardially with fixative containing 4 % PFA and 0.1–0.2 % glutaraldehyde (n = 3). Brainstems were removed and postfixed overnight in the same perfusate. Coronal brainstem sections were cut at 50 μm and collected into PBS. Sections were incubated in 50 % ethanol for 30 min to aid antibody penetration, before freeze fracturing in liquid nitrogen. Freeze-fractured sections were incubated in Guinea pig anti-VGLUT1 (1:10,000 in PBS, Millipore, UK) overnight at 4 °C and then in biotinylated donkey anti-Guinea pig secondaries (1:250 in PBS, Jackson ImmunoResearch, USA) for 4 h at room temperature, with three washes in PBS between each incubation. Sections were incubated in ExtrAvidin peroxidise (1:1,500 in PBS, Sigma, UK) overnight at 4 °C and visualised using diaminobenzidine as the chromogen. Stained sections were postfixed in 0.5 % osmium tetroxide (in 0.1 M PB) and dehydrated before embedding in Durcupan ACM resin (Fluka, Switzerland). Areas with suitable staining were prepared for electron microscopy and viewed on a Phillips CM10 transmission electron microscope. Negatives were digitised using an Epson 3200 Photo Perfection scanner.