Oligodendrocyte cultures were fixated with 4% PFA in PBS during 10 min. Subsequently 2% BSA and 0.1% saponin in PBS was used to block nonspecific binding. Plates were incubated with primary antibodies (rabbit‐anti‐Olig2, Merck Millipore AB9610; 1:1000 and mouse‐anti‐MBP, Biolegend SMI‐94, 1:1000) overnight at 4°C, washed with PBS, followed by incubation with alexafluor‐594 and ‐488 conjugated secondary antibodies (Life technologies; 1:1000) for 1 hr at room temperature. Cell nuclei were counterstained with Hoechst 33342 (Sigma) and wells were embedded in Fluorsave (Merck Millipore, 345789).
For each well, six adjacent fields were photographed (×10), starting at a fixed distance of the well edges. Olig2‐ and Hoechst‐positive cells were counted automatically using the analyze particles function in ImageJ v1.47. MBP area was determined using manual threshold analyses in ImageJ software v1.47. In order to reliably compare independent experiments, all results were normalized for the positive control (MCM + LPS; insert without MSCs/ 0 ng factor).
For each well, six adjacent fields were photographed (×10), starting at a fixed distance of the well edges. Olig2‐ and Hoechst‐positive cells were counted automatically using the analyze particles function in ImageJ v1.47. MBP area was determined using manual threshold analyses in ImageJ software v1.47. In order to reliably compare independent experiments, all results were normalized for the positive control (MCM + LPS; insert without MSCs/ 0 ng factor).