Glp 1r

For Western blot analysis, brains were homogenised (FastPrep FP120, Qbiogene, Illkirch, France) at 10% (weight/volume, w/v) in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl pH 8.0) and a subset of samples from comparable time points were digested with proteinase K (PK) (20 μg/ml) (Roche, Mannheim, Germany) for 1 h at 37 °C. Digestion was stopped by addition of 10× sample buffer and boiling for 10 min. Samples were analyzed by SDS-page (AnykD, Biorad, Hercules, USA), transferred to nitrocellulose membranes (0.2 μm pore size, BioRad) at 400 mA for 1 h, blocked for 1 h at room temperature in 5% milk powder in TBST buffer and incubated overnight at 4 °C with anti-PrP antibody Pom1 [50 (link)], Iba1 (Wako), Actin (Millipore), and GLP-1R (Santa Cruz) [63 (link)]. After washing and incubation for 1 h at room temperature with an HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:10.000 in blocking buffer), signals were detected with ECL femto reagent (Thermo Scientific) and visualized and quantified with a BioRad ChemiDoc imaging station and Biorad VersaDoc.

Brain tissues were fixed by perfusion with 4% paraformaldehyde (PFA), transferred to a 30% sucrose solution, and prepared in to 40 μm sections using a freezing microtome. The sections and neurons were blocked with BlockAid™ blocking solution (Invitrogen, Carlsbad, CA, USA) for 1 h and then immunostained using GLP-1R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Iba-1 (Abcam), GFAP (Cell signaling Technology, Danvers, MA, USA), MBP (Invitrogen), TBR1 (Abcam), CTIP2 (Abcam), SATB2 (Abcam), MAP2 (Cell signaling Technology, Millipore), and 4G8 (BioLegend, San Diego, CA, USA) antibodies at 4 ℃ overnight. The sections were then incubated with Alexa Flour 488 (Invitrogen) or Alexa Flour 594 (Invitrogen) conjugated secondary antibodies for 1 h at room temperature (RT). DAPI (Vector laboratories, Burlingame, CA, USA) was used for nucleus staining. The analysis was performed using a Zeiss LSM710 confocal microscope (Carl Zeiss, Göttingen, Germany). For immunohistochemistry, we used a Vectastain ABC IHC kit (Vector Laboratories, Burlingame, CA, USA) and Nikon Eclipse E600 microscope with a DS-Fi2 digital camera (Nikon, Melville, NY, USA).

Cultured SH-SY5Y cells or differentiated SH-SY5Y neurons were briefly washed in cold 1XPBS and fixed immediately with 4% paraformaldehyde in phosphate buffer saline (PBS) for 10 min. The formaldehyde fixed cells were incubated with a permeabilization buffer (0.5% Triton X-100, 100 mM glycine, 1% BSA, 0.7 mM EDTA) for 10 min on ice. The samples were then incubated with a blocking buffer (10% bovine serum, 0.01% sodium azide in 1X PBS) at 37 °C for 1 h or 4 °C overnight. Diluted primary antibodies (TUJ1, Sigma-Aldrich, 1:500; GLP-1R, Santa Cruz, 1:500) were incubated with samples for 1 h at 37 °C. The samples were then rinsed with wash buffer (0.05% Tween-20 in 1x PBS) three times for 5 min at room temperature. A fluorophore conjugated secondary antibody (Alexa series, Invitrogen, Carlsbad, MA, USA; 1:1000 dilution) was added and the samples were incubated for 1 h at 37 °C. Samples were rinsed briefly with wash buffer (3 × 5 min) and mounted with Prolong Gold containing DAPI (nuclear marker, Invitrogen). Images of immunostained neurons were captured using an Olympus FV10i confocal microscope system. The ImageJ (NIH) was used to quantitate the length of differentiated neurites.