Exploration of non‐radioactive alternative stains to uranyl acetate
Authors: Inoue Kanako , Muranaka Yoshinori , Park Pyoyun , Yasuda Hidehiro 
Published: December 2016

Uranyl acetate (UAc) has been generally used as a useful and stable staining reagent for ultrathin sections of biological materials in transmission electron microscopy. In Japan, however, the use and purchase of radioactive UAc is regulated by the government due to the safety problem. Therefore it is urgently necessary to find some non‐isotopic and non‐hazardous alternative electron stains. Taking into account the staining mechanism of UAc at the binding sites of biological tissues (Hayat, 2000), from among the heavy metals belonging to the lanthanoid group (non‐radioactive) we selected 3 reagents such as hafnium chloride (HfCl
4
), samarium chloride (SmCl
3
) and acetic acid gadolinium (GdAc) as the candidate alternative electron stains.

In order to evaluate precise stainability for these reagents, samples were prepared as follows: the resin blocks which embedded rat liver and rat small intestine mucosal were cut with ultramicrotome and a diamond knife. Serial ultrathin sections were mounted on copper grids coated with 0.5% Neoprene W in toluene. The sections were stained with 4% aqueous UAc and then with Sato's Pb. As to the candidate alternative electron stains, preparation processing was carried out in the same way. These stained serial sections were observed with a transmission electron microscope (TEM). For determination of relative TEM contrast ratios of organelles in the specimens, each of photo images from sections was given a numerical value of illumination intensity (black‐to‐white ratio) of average of 30 points for each target structure by using Photoshop software. The relative contrast ratio was expressed as density value of the target structure against a ratio of the value for the plain resin alone.

TEM observation showed that neither stain generated harmful effects such as uneven staining, contamination and cell disruption. The relative TEM contrast ratio analysis was indicated that HfCl
4
have a good staining effect for each biomembranes (nuclear membrane, cell membrane, organelle membrane), nucleolus, mitochondria, rough endoplasmic reticulum, glycogen and heterochromatin at the same level as UAc. Superior stainability of ribosome and mitochondria was obtained on SmCl
3
staining. GdAc exhibited good stainability for cytoskeletal filament such as actin filament in addition to organelles, biomembranes and cell components. From these results, GdAc could be excellent substitute for UAc for thin section staining. Furthermore, HfCl
4
was helpful for carbohydrate staining, and SmCl
3
was for protein substance, which indicated that these stains have different staining mechanism. Moreover, we examined the stain permeability because we frequently analysis semi‐thin section samples with ultra‐high voltage electron microscope. As the results, good stainability were obtained in the sections treated with HfCl
4
and GdAc, respectively. To provide a stain reagent properly depending on the purpose, further development would be required from now on. Additionally, to assess general versatility, we should test the stainability with various samples such as plant, fungi and also material samples.