Immunofluorescence Staining of Myelin Segments

Cells on coverslips or dishes were normally fixed with 4% paraformaldehyde (PFA). In all, 4% PFA followed by 100% cold methanol was used for myelin segment staining47 (link)48 (link)49 (link). Cells were permeabilized with phosphate-buffered saline (PBS) containing 0.1% Tween-20 or 0.1% Triton X-100, blocked with Blocking One reagent, and incubated first with primary antibodies and then with fluorescence-labelled secondary antibodies. The coverslips or dishes were mounted with Vectashield reagent with DAPI (Vector Laboratories, Burlingame, CA, USA). The fluorescence images were captured with a DMI4000B fluorescence microscope system (Leica, Wetzlar, Germany) and analysed with AF6000 software (Leica). At least three experiments were carried out under each condition and representative photographs are shown in the figures.