184 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Figure 1. Freeze-fractured lanolin showing emulsified transepidermal water. base of the desiccator held a saturated solution of calcium chloride dihydrate to provide a relative humidity of 38% within the chamber. The desiccator was kept in a constant- temperature room at 37øC. Treated skin was sampled after 16 hours. At the end of the treatment period, skin was removed from the holders, and small, roughly cubical pieces about 1.5 mm per side were cut for fixation by the standard operating procedure as follows: Primary fixation: Buffer wash: Secondary fixation: Wash: Dehydration: Infiltration: Embedding: Sectioning: Staining: Observations and microscopy: Freeze-substitution medium: 3% Glutaraldehyde solution in phosphate buffer 0.1 M Mixed phosphate buffer at pH 7.2 1% Solution of osmium tetroxide in buffer Distilled water Ethanol series 50%, 70%, 90%, 100% (twice) Propylene oxide 50:50 Propylene oxide: resin 100% Resin at 37øC 100% Resin 100% Resin in flat embedding molds Polymerized at 65øC By Reichert OMU2 ultramicrotome Some sections unstained, others stained in uranyl acetate and lead citrate solutions JEOL 1200 EX transmission electron microscope Methyl alcohol (100%) 100 ml

MOISTURIZING FUNCTION OF LANOLIN 185 Uranyl acetate 0.5 g Osmium tetroxide 1.0 g Glutaraldehyde stock solution (50%) 6.0 ml Results. Figure 2 is a section of the outer layers of stratum corneum showing an excess of fanolin (grey) remaining on the surface (bottom of picture) after 16 hours, and also some fanolin (similarly grey) that has penetrated into intercellular spaces down to the third or fourth corneocyte. A dark line following the edge of some corneocytes suggests a particularly high concentration of lanolin (or at least lead marker) at those points, as though lanolin had become incorporated in the trilaminar outer membranes of corneo- cytes that in places had become detached from the cell. Most other intercellular lipids appear to have been lost during specimen processing. The surface lanolin is seen to contain some very electron-opaque particles, which are believed to be, in part, a residual impurity of unreacted lead nitrate in the doped lanolin. An examination of a thin film of this lanolin by electron microscopy showed similar small, very dense clumps. Figure 3 is of another skin section at greater magnification and shows even more clearly an accumulation of lanolin (and/or lead marker) in laminar structures. The largest such feature is particularly ionteresting since is shows a distinct triple layer consistinog of a central band about 200 A thick sandwiched between two narrower (80 to 100 A) bands that are more electron-opaque. This structure is in accordance with the findings of Swanbeck (9), who proposed the existence of fibrous protein layers sur- rounded by lipid bilayers, which, in our own case, have apparently been infiltrated by I #L. ??LAN. 16H. i 9!6552 88.ekU XI5K 5•0n• Figure 2. Lanolin on skin surface (bottom of picture) showing penetration into intercellular spaces and concentration at cell surfaces.