A protocol for preparation of chromosome spread and processing for transmission electron microscopy

Abstract

Transmission electron microscopic (TEM) observation of human chromosome ultrastructure may provide useful information on various abnormalities. Due to the difficulties in detaching metaphase chromosome spreads from the glass slide, this approach has been restricted. We introduce a simplified protocol in which the metaphase chromosome spreads are made on a flexible thermoplastic membrane (Aclar film) glued to the routine microscopic glass slides. The chromosome spreads are then impregnated with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. After polymerization, the epoxy resin sheet is easily detached from the Aclar film and all chromosome spreads retain on the resin sheet. Each chromosome spread is identified under a light microscope, demarcated, trimmed then attached to a pre-polymerized blank epoxy resin block. Ultrathin sections are cut and observed under a transmission electron microscope. Application of this method to chromosome research is suggested to provide useful information on the chromosome morphology and ultrastructure in relation to various conditions and/or function.