New Staining Method for Electron Microscope Specimens Safer and Cost-Effective

The electron microscopic staining technique for biological specimens using uranyl acetate (UA) was first reported in 1958 and has been used in electron microscopy facilities worldwide because of its simplicity and optimal staining results. In recent years, however, international regulations on the use, acquisition, storage, and disposal of uranium compounds have become increasingly strict because uranium is used as a nuclear material for weapons. Against this backdrop, alternative staining methods have long been long awaited in the field of biological research, and several have been proposed, but none has been an effective alternative. In this context, alternative staining methods have long been expected in the bio-medical electron microscopic research field, and several have been proposed, though none has been an effective alternative. Now, researchers have developed a new staining method for electron microscope specimens based on double staining with hematoxylin, which is widely used as a staining agent for light microscopy, and lead solution. The new method is expected to replace the conventional method of double staining with uranium acetate and lead solution and is a better choice in terms of safety, cost, and ease of handling.

To develop a safe and easy-to-handle alternative staining method to UA for ultrathin sectioning in electron microscopy, researchers at Tokyo University of Technology (Tokyo, Japan) examined various commercially available dyes for light microscopy. The researchers found that double staining with Mayer's hematoxylin (MH), which is commonly used as a staining agent in light microscopy, and lead solution (Pb), exhibited staining properties equivalent to those of conventional electron microscopic double staining with UA and Pb in various tissues and cells.

Other cell organelles, such as nuclear chromatin, plasma membrane structures, ribosomes, glycogen, lipid droplets, cell adhesion apparatus, and cytoskeletal system(s), were stained with high contrast using the MH staining method. Plasma membrane staining in all samples was also satisfactory. Backscattered electron images of 200 nm semi-thin sections of mouse kidney observed by a field emission scanning electron microscope also showed that the MH and Pb staining techniques provided a wide-area and high-quality image of the renal cortical tubules and the renal glomerulus.

MH is a dye solution widely used for staining paraffin sections of diagnostic clinical specimens, and has the advantages of low cost, stable supply as a commercial product, and high safety of waste solution. On the other hand, the International Atomic Energy Agency's "International Basic Safety Standards for Protection against Ionizing Radiation and for the Safety of Radiation Sources" (BSS) has established specific exemption levels, and new regulations for radioactive materials are being developed internationally through legislation. Based on the results of this study, double staining with MH and Pb is expected to become a useful alternative to the staining method using radioactive UA in terms of reagent purchase, handling, use, storage, and liquid waste treatment.

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Tokyo University of Technology