Anti glp 1r

Western blot tests were performed as previously reported (Sozen et al., 2009 (link)). Briefly, the whole left hemisphere was isolated and collected 24 hours after SAH (n=6 per group). Equal amounts of protein samples were loaded onto each lane of SDS-PAGE gel. After electrophoresis, the samples were transferred onto a nitrocellulose membrane, which was blocked with a blocking solution for 2 hours. The membrane was incubated at 4 °C overnight with the following primary antibody: anti-Bax (1:500, NBP1-28566, Novus Biologicals, CO, USA), anti-Bcl-2 (1:1000, ab59348), anti-PI3K (phospho Y607) (1:1000, ab182651) (Abcam, Cambridge, MA, USA), anti-Akt (1:1000, #9272), anti-phospho-Akt (Ser473) (1:1000, #9271), anti-Bcl-xl (1:1000, #2764) (Cell signaling, Boston, MA, USA), anti-GLP-1R (1:200, sc-390774), and anti-β-actin (1:5000, I-19) (Santa Cruz Biotechnology Inc., Texas, USA). Appropriate secondary antibodies (1:5000, Santa Cruz Biotechnology Inc., and anti-rabbit 2839792, EMD Millipore Corporation, MA, USA) were selected to incubate with the membrane for 2 hours in room temperature. The bands were detected with an ECL regents (Amersham Biosciences UK Ltd, PA, USA) and quantified by densitometry with Image J software (Image J 1.4, NIH, USA). β-actin was blotted on the same membrane as loading control.

The brain samples for immunohistochemistry staining were prepared following previously reported procedures (Enkhjargal et al., 2016 ). Briefly, animals were deeply anesthetized at 24 hours after SAH and intracardially perfused with 60 mL ice-cold PBS followed by 60 mL of 10% paraformaldehyde through the upper part of the body. The whole brain was collected and fixed in 10% paraformaldehyde for 24 hours followed by 30% sucrose for three days. After being frozen at −80 °C, the brains were cut into 10 μm thick coronal sections on a cryostat (LM3050S; Leica Microsystems, Bannockburn, III, Germany). Double fluorescence labeling was processed as previously described (Enkhjargal et al., 2014 (link)). Brain sections were incubated overnight at 4 °C with primary antibody including anti-GFAP (1:500, ab53554), anti-NeuN (1:500, ab177487), anti-iba-1 (1:500, ab107159) (Abcam, Cambridge, MA, USA), and anti-GLP-1R (1:50, sc-390774, Santa Cruz Biotechnology Inc.). Corresponding secondary antibodies (1:500, Jackson Immunoresearch, West Grove, PA, USA) were incubated with the sections at room temperature for 2 hours. The sections were visualized and photographed with a fluorescence microscope (Leica Microsystems, Germany).