Azure c300 gel imager

Cells and decellularized spinal cords were harvested in RIPA buffer (10 mM Tris-HCl pH 8.0, 140mM NaCl, 1mM EDTA, 0.1 % SDS), supplemented with 1 % Triton X-100 (Triton, Sigma Aldrich), 1mM phenylmethylsulphonyl fluoride (DOT Scientific), and protease inhibitors (Millipore). For WB analysis lysates were sonicated and protein extracts were separated by SDS-PAGE followed by electrotransfer to a nitrocellulose membrane (Bio-Rad). For dot-blot analysis, 10 μg of protein was loaded through a 96 well Acrylic Dot-Blot System (Whatman, GE Healthcare Life Sciences) directly onto a nitrocellulose membrane. The membranes were blocked in Tris-buffered saline (TBS, Bio-Rad) + 5 % non-fat dry milk (Bio-rad) and then incubated overnight at 4 °C with primary antibodies. Primary antibodies were diluted in TBS + 0.1 % Tween + 5 % BSA (Sigma Aldrich). After several washes in TBS + 0.1 % Tween, membranes were incubated with their corresponding secondary HRP-conjugated antibodies (ThermoFisher). A radiance bioluminescent ECL substrate kits (Radiance ECL, Azure Biosystems or Pierce ECL Western Blotting Substrate, Thermofisher) was used to detect protein signals through Azure c300 Gel Imager (Azure) or Image Quant LAS4000 mini-imager (General Electric) on the automatic setting. Densitometry analysis for the bands was performed using Fiji software (ImageJ, NIH Image).