Preparation of Human Scalp HFs for TEM

Human scalp HFs were fixed in a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer for 2 h at room temperature and then processed for TEM as described in the literature [47 (link)]. Briefly, the samples have been washed in cacodylate buffer (0.1 M, pH 7.4), postfixed for 2 h with 1% osmium tetroxide in the same buffer, extensively washed again, and then incubated overnight in a 0.5% uranyl acetate aqueous solution in the dark. After washing, the sections have been dehydrated in a graded alcohol series, and after a final dehydration in 100% propylene oxide, they have been infiltrated with low viscosity Spurr resin overnight and polymerized for 48 h at 65 °C. Sections of about 70 nm were cut with a diamond knife (DIATOME) on a Leica EM UC6 ultramicrotome. Bright field TEM images have been collected with a Schottky field-emission gun FEI Tecnai G2 F20 (FEI, USA) transmission electron microscope operating at an acceleration voltage of 200 kV and equipped with a 2k × 2k Gatan Ultrascan (Gatan, USA) charge coupled device (CCD).