Investigation of substitute mordants in Gomori’s one-step trichrome stain

Abstract

Currently, the method of fixing tissue sections for histopathological analysis is to use a formalin solution alone; thus, pretreatment of sections with mordants containing potassium dichromate or Bouin’s solution is required when performing collagen staining. However, a review is urgently needed from the viewpoint of toxicity of these chemicals. In this study, we investigated the usefulness of substitute mordants in Gomori’s one-step trichrome staining. Firstly, the sections were pretreated with substitute mordants of Bouin’s solution, saturated picric acid solution, decalcifying solution A (Plank Rychlo method), and 0.5% potassium permanganate solution at 25°C for 30 min or at 60°C for 60 min. Secondly, the sections were stained in accordance with the original Gomori’s one-step trichrome staining method. Lastly, the stainability was investigated by microscopy and RGB/Br (Red-Green-Blue/Brightness) analysis. The B/Br value of the saturated picric acid solution at 60°C for 60 min was significantly lower than that of commercially available mordants containing dichromic acid and decalcifying solution A. In other words, this mordant suppressed blue costaining in the cytoplasm, dyed the cytoplasm more vividly red than other mordants, and improved the contrast with blue in collagen fibers. On the other hand, pretreatment of the sections with 0.5% potassium permanganate solution resulted in poor staining quality. Whereas chromic acid and formaldehyde are carcinogenic, picric acid is not. Therefore, in this study, it was demonstrated that pretreating the sections with a saturated picric acid solution can be as effective as the original Gomori’s one-step trichrome staining method.