680 rd secondaries

15-20 μg of protein were separated on 4-12% Bis-Tris NuPAGE gels (Thermo-Fisher) according to manufactures instructions. Gels were either stained for total protein using 0.3% w/v brilliant blue-G (Sigma) in 40% v/v methanol and 7% v/v glacial acetic acid overnight. Destaining was done with several washes in 40% v/v methanol and 7% v/v glacial acetic. For Western blotting, proteins in gels were transferred onto PVDF membranes (Millipore) using Bolt transfer buffer (Thermo-Fisher) for 1 hour at 15 V constant voltage. Membranes were blocked with Li-Cor Blocking Buffer (Li-Cor) for 1 hour at room temperature on a platform shaker. Membranes were incubated overnight in primary antibodies (1:2000 dilution) rabbit-anti-Mbp or mouse-anti-Mog (Cell Signaling), rabbit-anti Neurofilament heavy or mouse anti-synaptophysin (Sigma) in 50% Li-Cor Blocking Buffer in tris buffered saline with tween-20 (0.001% v/v) (TBS-T). Membranes were washed three times with TBS-T and Li-Cor 680-RD secondaries (1:5000) (Li-Cor) applied in 50% v/v Li-Cor Blocking Buffer in TBS-T for 1 hour at room temperature. Membranes were three times in TBS-T and imaged on a Li-Cor Odyssey (Li-Cor).