Fluorescent Visualization of GLP-1R Expression in Brain Vasculature

Mice expressing fluorescently tagged GLP-1R (Glp1r-cre/tdRFP) [16 (link), 71 (link)] were used to determine the expression of the receptor by the brain blood vessels. The animals were terminally anaesthetised with sodium pentobarbital overdose (200 mg kg⁻¹; i.p.) and perfused through the heart with 0.01 M phosphate-buffered saline. The brains were removed and fixed overnight in 4% paraformaldehyde. Free-floating sections (20 µm thickness) were incubated (overnight at 4 °C) with anti-DsRed antibody (1:500) (Takara Bio) to detect GLP-1R expressing cells and anti-smooth muscle actin (SMA) antibody conjugated to FITC (1:350) (Sigma) for the detection of arteries and arterioles. Incubation with anti-rabbit AlexaFluor 568 (1:200, 2 h) was performed on the next day, followed by endothelial cell labelling using biotinylated lectin (1:250, 30 min) (Vector Biolabs), visualised using AMCA streptavidin (1:100, 1 h) (Vector Biolabs). Tiled images of coronal cortical cross-sections were obtained using Zeiss LSM 800 confocal microscope.