The ultraplaning technique for SEM specimen preparation

The technique of ultraplaning tissue consists of impregnating tissues with osmium tetroxide and thiocarbohydrazide (OTOTO), uranyl acetate and embedding in hard polyethylene glycol followed by planing a ultrasmooth surface with glass knives in an ultramicrotome. Subsequently, the tissue is stained with lead citrate, dehydrated, critical point dried and examined uncoated in an SEM. High resolution cut-surface and surface details are visible, permitting recognition of changes in surface contour and changes in intracellular organelles. An alternate procedure using celloidin-paraffin embedding consists of the following. OTOTO treated tissues are infiltrated with celloidin, hardened in chloroform, and infiltrated in paraffin. After the block is ultraplaned, the paraffin is removed with xylene. The tissues are hydrated and processed as with the polyethylene glycol procedure. This method has the advantage of holding fragile specimens together during the processing.