Anti vgat antibody

First, the sections were removed from the PBS and placed in a fresh solution of 1% sodium borohydride in 0.1 M PBS for 30 minutes at room temperature (RT). They were then incubated for 30 minutes at RT in 1% bovine serum albumin and 0.1% gelatin in PBS at pH 7.4 (PBS-BSA). Next, sections were incubated for 48 hours with the following primary antibodies in blocking solution: rabbit anti-p-Syn I antibody (ser-62/67 and ser-553; 1∶1000 dilution; Santa Cruz Biotechnology), anti-VGLUT1 antibody (BNPI; 1∶1000 dilution; Santa Cruz Biotechnology), and anti-VGAT antibody (1∶1000 dilution; Merck Millipore). Subsequently, the sections were incubated with secondary antibodies (Alexa Fluor488 FluoroNanogold,Nanoprobes, NY, USA) diluted 1∶100 in PBS-BSA with 1% goat serum for 1 hour at RT, and then they were post-fixed with 1% glutaraldehyde in PBS for 10 minutes. Finally, silver enhancement with a HQ silver EM intensification kit (Nanoprobes) was performed for 6 minutes and then the sections were rinsed three times in deionized water for 10 min.

Proteins (30–50 µg) from each sample were fractionated using 8% sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Merk Millipore, Billerica, MA, USA). The membrane was blocked in 5% low fat milk powder in PBS for 1 hour at 4°C and probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1∶10000 dilution; Santa Cruz Biotechnology) rabbit polyclonal primary antibody, anti-p-Syn I (ser-62/67, ser-553, and ser-603; 1∶1000 dilution, Santa Cruz Biotechnology), anti-VGAT antibody (1∶1000 dilution; Merck Millipore), anti- VGLUT1 antibody (BNPI; 1∶1000 dilution; Santa Cruz Biotechnology).Bands were visualized by an enhanced chemiluminescence detection system using FluorChem FC2 (Alpha Innotech, USA) and quantified by Photoshop (Adobe software). The intensities of band of interest were expressed relative to the GAPDH intensities from the same sample.