051 Visualisation of hair cortex filaments in situ by transmission electron microscopy: A simpler procedure revisited.

G.E. Rogers, South Australian Research and Development, Glenside, Adelaide South Australia, Australia

The standard procedure for introducing contrast into thin sections of hard alpha (and also beta keratins) for electron microscopic studies was introduced over forty years ago (GE Rogers, J. Ultrastruct.Res.2, 309 1959). For example, hairs collected in a bundle are submitted to partial reduction of disulphide bonds by a thiol reagent (thioglycollic acid or â-mercaptoethanol) at pH5 followed by bulk staining with osmium tetroxide and resin embedding. Thin sections are then stained with uranyl acetate and Reynold’s lead citrate. This procedure produces excellent visualisation at high resolution of all of the major components of a variety of hairs. The standard method takes about three days to prepare specimens. A more rapid procedure was briefly described for visualising keratin filaments in thin sections of porcupine quill tip (GE Rogers, & BK Filshie, Proceedings of 5th Int.Conf. Electron Microsc. Philadelphia, 1962, 2, O-2) that dispensed with the reduction-osmium step and used 1% potassium permanganate at pH 6.0 for 30 min on thin sections of untreated resin-embedded fibres followed by lead staining. It has now been observed that the organisation of the keratin intermediate filaments in cross-sections, cell membrane complex and internal organisation in the cuticle of human and mouse hairs can be rapidly visualised as follows:- Small bundles of say 10-20 hairs depending on diameter, without dehydration or other pretreatment, are directly resin-embedded with orientation and thin sections cut and floated on 1% potassium permanganate in 0.1M phosphate buffer pH6 for only 10 min. and the dried grids examined in the EM. If necessary, contrast can be increased by 5 min staining with 4% aqueous uranyl acetate. The processing mainly involves the overnight curing of the resin. The mechanism probably depends on oxidation of disulfide bonds and deposition of a manganeseprotein complex in the interfilamentous matrix. The uranyl cation probably binds to sulfonate groups produced by this oxidation. The procedure produces some debris on the grid but its identity has not been established.