Detection of Histamine in Rat Mast Cell Granules by Orcein-Water Blue Stain

Abstract

A new method of staining histamine in rat mast cell granules in formalin fixed paraffin section was developed.Mast cells of several organs of formalin fixed paraffin sections were stained well in red with orcein-water blue as well as in purple with toluidine blue.But only mast cells in dermis showed a different appearance in orcein-water blue stain and toluidine blue stain. The results of an electrophoretic study, diamine oxidase treatment and compound 48/80 treatment of rat lead to the conclusion that the substance stained in red with orcein may be histamine.It was shown that histamine in rat mast cell granule is fixed even by formalin, a finding contrary to previous studies.The mast cell contains many substances in its granules. Histamine is one of the principal substances in granules. The histochemical demonstration of histamine is significant in the study of mast cells. It has been considered that histamine in tissue easily dissolves out by formalin fixation, thus known staining methods require a complicated technique in fixation or staining because of the solubility of histamine (3, 4, 7).In the present study, we obtained evidence that histamine can be detected even in formalin fixation. This article reports on the effectiveness of orcein in staining histamine in formalin fixed paraffin sections. MATERIALS AND METHODSAdult male Wister rats weighing 200-300 g were anesthetized with ether and exanguinated by cutting the carotid arteries, except when otherwise specified. The tongue, skin, lung, stomach and ileum were removed and fixed overnight or longer in 10% formalin. All specimens were embedded in paraffin, cut in 5µm sections with the microtome, deparaffinized and used for staining.Staining methods Orcein-water blue stain :The orcein-water blue solution was made by dissolving 0.98 g of orcein (Wako Chem. Co., Ltd., Japan) in 60 ml of 100% ethyl alcohol to which was added 1 ml of concentrated HCl mixed with 0.02 g of water blue (also Wako Co.) dissolved in 40 ml of distilled water (2). The solution was stirred thoroughly, filtrated and used for staining. Sections were treated with orcein-water blue solution for 10 min and differentiated by 60% alcohol with 1 % concentrated HCI, dehydrated, cleared and mounted.

Morphological Features of Doxorubicin-Resistant Walker 256 Carcinosarcoma and Response of Mast Cells

“…Histamine in MCs was detected in the sections stained with solution of Water Blue -Orcein [19]. The results of cytochemical reaction were expressed as a percentage of positive cells [20].…”