Biotinylated donkey anti sheep antibody

Sections were dewaxed, hydrated, and incubated in 0.1% pronase in TBS-T buffer for 10 min. Sections were then blocked with avidin-biotin (004303, Thermo Fisher Scientific, Waltham, MA) prior to incubation with 5 μg/ml rabbit anti-GLP-1R antibody (7F38, Novo Nordisk A/S, Denmark) for 1 h at RT. Following washing in TBS-T, sections were incubated with biotinylated donkey anti-rabbit antibody (1:200, 711-165-152, Jackson ImmunoResearch, Cambridgeshire, UK) for 30 min. Sections were washed in TBS-T and signal amplification was performed with ABC elite kit (PK6100, Vector Laboratories, Burlingame, CA) followed by incubation in 1:50 TSA-Flourscein-Cy2 (SAT701001EA, Perkin Elmer, Waltham, MA) for 40 min. Next, the sections were incubated in TEG buffer (pH 9.0) for 15 min, washed in TBS-T, blocked with avidin-biotin, and incubated in TNB for 30 min prior to incubation with 0.2 mg/ml sheep-anti GFRAL antibody (PA547769, Thermo Fisher Scientific) over night at 4 °C. Following washing in TBS-T, sections were incubated in 1:1000 biotinylated donkey anti-sheep antibody (713-065-147, Jackson Immuno Research) for 30 min at RT. Signal amplification was performed with 1:100 TSA Tyramide-Cy3 (SAT704A001EA, Perkin Elmer). Sections were washed in dH2O and mounted with fluorescent mounting media (S3023, Dako). Confocal images were acquired using the Leica Confocal Imaging System TCS SP8.