EXTRACELLULAR material in a variety of connective tissues is coloured by Sudan black dissolved in acetone. This material exhibits properties characteristic of bound lipid (MELCHER, 1966, 1967a,b). The extent and intensity of extracellular sudanophilia is greatly increased by extraction of unfixed sections in acidified chloroform : methanol (Ac.C:M), suggesting that further bound lipid is unmasked to staining by this procedure. Similar extraction after fixation is ineffective (unpublished data) so that, if hard tissues are to be examined for sudanophilic bound lipids by these methods, they must be demineralised without prior fixation. In this investigation, sudanophilic material was sought in sections of teeth and their supporting connective tissues which had been demineralised, unfixed, in ethylenediamine-tetra-acetic acid (EDTA). However, since SHAPIRO, WUTHIER and IRVING (1966) and SHAPIRO and WUTHIER (1966) have shown that exposure of dental hard and soft tissues to EDTA enhances the subsequent extraction of some of the constituent lipids, it is conceivable that EDTA could unmask bound lipids to histological staining. To test this, the consequences of immersing skin in EDTA were also examined. Mice (A2G), weighing 25-30 gs, were sacrificed by cervical dislocation, and the molar segments of the mandibles and two pieces of dorsal skin were resected and treated as follows: (a) One piece of skin was frozen immediately. Sections were cut at right angles to its surface in a cryostat, mounted on slides and stored at 25°C. (b) The hard tissues and the second piece of skin were immersed in 10% (w/v) EDTA brought to pH 74 with N/2 sodium hydroxide and kept either at room temperature or at 4°C. After demineralisation (about 4 days at room temperature and 8 days at 4”C), the tissues were washed in running tap water for 24 hr. The skin was then frozen and sectioned as described above. The hard tissues, on the other hand, were first embedded in gelatin and then frozen, after which cryostat sections were cut approximately parallel to the occlusal surfaces of the teeth and mounted on slides. Some mounted sections of both hard tissues and skin were extracted unfixed in either: (a) Ac.C :M (chloroform : methanol : concentrated hydrochloric acid, 2 : 1: l/300 v/v/v) at room temperature for periods varying from 5 min to 48 hr.
