Optiblot ecl detection kit

Brain tissue samples were lysed with RIPA-buffer (Thermo Scientific, Waltham, MA, USA), containing “Complete” Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA), and was sonicated and centrifuged for 15 min at 14,000× g. The protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). An amount of 1μg of total proteins were dot-blotted onto PVDF membranes at a volume of 1 μL. The membranes were blocked with 5% non-fat milk for 1 h and incubated with mouse anti-heparan sulfate primary antibody (1:500 Millipore, Burlington, MA, USA) overnight at 4 °C, followed by secondary peroxidase-conjugated antibodies goat anti-Mouse IgG (Abcam, Cambridge, UK) for 1 h at RT. GAGs were detected using an Optiblot ECL Detection Kit (Abcam, Cambridge, UK), according to the manufacturer’s instructions. Blots were imaged using ChemiDoc (BioRad, Hercules, CA, USA) and analyzed semi-quantitatively using ImageJ 1.52 software.

Brain tissue samples were lysed with RIPA-buffer (Thermo Scientific, Waltham, MA, USA) containing “Complete” Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA), sonicated using a Microson XL-2000 Ultrasonic liquid processor (Qsonica, Newtown, CT, USA) at 15 µm amplitude (45 W) and centrifuged for 15 min at 14,000 gat + 4 °C. The protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Quantities of 1 μg of total proteins were dot-blotted onto PVDF membranes (Millipore, Burlington, MA, USA in a volume of 1μL. The membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with mouse anti-CS primary antibody (Sigma-Aldrich C8035, CS-56, 1:500) or anti-HS antibody (Millipore, clone T320.11, 1:500) diluted in 1% non-fat milk in PBST overnight at 4 °C followed by the secondary peroxidase-conjugated antibody goat anti-Mouse IgG (Abcam, Cambridge, UK) for 1 h at room temperature. GAGs were detected with an Optiblot ECL Detection Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Blots were imaged using ChemiDoc (BioRad, Hercules, CA, USA) and analyzed semi-quantitatively using ImageLab 6.0.1 (BioRad, Hercules, CA, USA) software, the chemiluminescent signal was normalized to total protein content according to Coomassie staining.

Brain tissue samples were lysed with RIPA-buffer (Thermo Scientific, USA), containing “Complete” Protease Inhibitor Cocktail (Roche, USA), sonicated, and centrifuged for 15 min at 14,000g. The protein concentration was quantified using Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). 1 μg of total proteins were dot-blotted onto PVDF membranes in a volume of 1 μl. The membranes were blocked with 5% non-fat milk for 1 h and incubated with mouse anti-CS primary antibody (1:500, CS-56, C8035, Sigma-Aldrich, USA) overnight at 4°C followed by secondary peroxidase-conjugated antibodies goat anti-Mouse IgG (Abcam, UK) for 1 h at RT. GAGs were detected with an Optiblot ECL Detection Kit (Abcam, UK) according to the manufacturer’s instructions. Blots were imaged using ChemiDoc (BioRad, USA) and analyzed semi-quantitatively using ImageJ 1.52 software.