Osmium maceration for human bioptic specimens

We have further modified the aldehyde/OsO 4 /dimethylsulfoxide (DMSO)/freeze cracking/DMSO/OsO 4 / DMSO maceration method, in order to render it more suitable to the analytical study of normal and pathological human bioptic samples. The new technique, which has been made easier and quicker, allows the visualization not only of cytoplasmic organelles but also of internal surfaces. The changes consist of the use of 1% OsO 4 -1.25% K 4 Fe(CN) 6 as a secondary fixative and in the sectioning of tissues at room temperature by a chopper. The OsO 4 ferrocyanide mixture, by binding osmium to the tissue, allows the omission of further treatment with OsO 4 and tannic acid. It also allows the storage of specimens in phosphate buffered saline at 4°C for several days before the start of the maceration. Since internal structures are exposed by cutting and not by freeze fracturing, the treatment with DMSO becomes unnecessary. Furthermore, 44 hours at 25°C is enough to macerate specimens with 0.1% OsO 4 thereby reducing the total time for preparation to 48 hours. By shaking the specimens during or after maceration, we were able to remove organelles and to expose the inner surface of the plasmalemma and its special structures (e.g., folds, intercellular canaliculi, and cellular junctions). A further result is the visualization, in hepatic needle biopsies, of pathological features of possible diagnostic value.