Odyssey clx scanner

For Western blot analysis, neuron/glia co‐cultures were grown for 12 days on 6 cm culture dishes plated with 500,000 cells. Cultures were washed two times with ice‐cold PBS, harvested, and homogenized by sonication. The samples were incubated with Laemmli sample buffer (Bio‐Rad) for 1 h at 37°C and aliquots containing 25 μg protein were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) using mini‐gels (Mini Protean 3; Bio‐Rad). Proteins were transferred onto nitrocellulose membranes in a semi‐dry transfer cell (Trans‐Blot SD; Bio‐Rad) at 15 V for 75 min. After blotting, the transferred total proteins were stained with REVERT 700 Total Protein Stain (LI‐COR Biosciences) and detected by the ODYSSEY CLx scanner (LI‐COR Biosciences). After destaining, the blots were blocked for 1 h in PBS containing 5% nonfat dry milk at room temperature, followed by incubation with primary antibody overnight at 4°C in PBS containing 5% nonfat dry milk. The blots were then washed five times for 5 min with TBST and incubated with secondary antibodies for 1 h at room temperature. The blot was washed again with TBST and immunoreactivity was detected by the ODYSSEY CLx scanner (LI‐COR Biosciences). Immunoreactivity was quantified with the Image Studio software (LI‐COR Biosciences) and normalized to total protein in the corresponding lanes.