2019 Volume 68 Issue 1 Pages 117-123
Berlin blue staining is a method of detecting trivalent ions in a specimen. The staining solution must be prepared immediately prior to use. We examined the effects of different storage conditions on the dyeability and changes in absorbance of the staining solution to clarify its convenient storage conditions. The storage conditions were at room temperature or 4°C and without light, and under cryopreservation (−80 and −20°C). The absorbance of the cryopreserved solution was maintained at its initial value, but those of the other solutions increased depending on the storage conditions. The cryopreserved solution showed no change in color tone and staining strength up to 1 year. It caused no false positive results. However, for the solutions that were not cryopreserved, their color tone changed from blue to green, their staining strength decreased, and they caused an increase in the number of false positive results. The freezing speed and cryopreservation temperature did not affect the results. The absorbance changes showed that trivalent ferric salts were generated over time and reacted with potassium ferrocyanide, then free iron ferrocyanide (Berlin blue) was generated in the solutions without cryopreservation. Thus, it seems very likely that the concentration of potassium ferrocyanide, which binds to iron ions in tissues, decreased, and therefore the dyeability of the solution weakened. The change in color tone from blue to green might be due to the change in the molecular weight of the Berlin blue dye. When the solution was frozen, these reactions ceased so that there was no change in absorbance or dyeability. These results indicate that the cryopreservation of the staining solution is convenient and useful for Berlin blue staining.
