A NEW METHOD FOR FLAT-EMBEDDING LARGE NATIVE CRYOSTAT SECTIONS FOR TARGETING SMALL PRENEOPLASTIC LESIONS IN COMPARATIVE ULTRASTRUCTURAL ANDULTRACYTOCHEMICAL INVESTIGATIONS

Ultrastructural studies of rare and small cellular lesions in patholog ically altered tissue are difficult to perform, by applying convention al electron microscopic preparation. The search for lesions, often con sisting of only a few cells in randomly obtained small specimen blocks , is time consuming and often without success. The methodological requ irements for comparative enzyme cytochemical and morphological studies , i.e., preservation of both enzyme activity and ultrastructure, are d ivergent. By processing large native cryostat sections for electron mi croscopy, small preneoplastic focal lesions were successfully targeted ir liver and kidney. Glucose-6-phosphatase,; alkaline phosphatase, ac id phosphatase, catalase, and cytochrome c oxidase activities were dis tinctly localized to endoplasmic reticulum, canalicular membrane, lyso somes, peroxisomes, and mitochondria, respectively, in the morphologic ally altered cells. Fixation of serial cryostat sections and enzyme re actions were both carried out through a semipermeable membrane except those for cytochrome c oxidase, which was demonstrated after fixation through the membrane by floating the section in incubation medium cont aining cytochrome c. Thereafter, the sections were flat embedded and p olymerized between epoxy resin disks and aluminum dishes fitting exact ly together. The objects of interest were identified in the light micr oscope, cut out, and reembedded in reversed gelatine capsules. By usin g this technique an ultrastructural preservation was achieved similar to that seen after immersion fixation. The enzyme activities were clea rly localized without diffusion of the reaction product or unspecific deposits. The procedure permits precise targeting and complex studies of rare and small lesions, and opens new perspectives for the use of c ryo-preserved tissue.