Fixation and Fixatives
ALCIAN BLUE
RATIONALE FOR USE
Alcian blue is a water soluble, amphoteric copper phthalocyanine, which is most often used as a basic dye. In general, alcian blue has an affinity for sulfated and carboxylated groups of acid mucopolysaccharides, but the specific group demonstrated is dependent on the dye solution pH.
RECOMMENDED FIXATIVES
10% neutral buffered formalin and Bouin’s fixative.
AVOID Chromate Fixatives
MODE OF ACTION (1, 2, 3)
Alcian blue binds electrostatically with the acidic groups in the mucopolysaccharide molecule. The components to be demonstrated are intensely stained if the dyer solution is used at the specific pH at which the reactive groups are fully ionized. To achieve full ionization of the reactive groups, some alcian blue methods begin with a rinsing of sections in the appropriate pH solution prior to staining with the dye solution. In general, strongly sulfated mucins react more consistently at low pH levels. Above a pH of 1.0 their reactions are variable. At a pH of 0.2 only strongly sulfated mucosubstances will be demonstrated, while weak sulfated mucins stain well between a pH of 1.0 - 2.5, and often below 1.0. Carboxylated mucins react with alcian blue at pH 2.5.
Alcian blue produces an intense greenish-blue or teal coloration. To ensure this colour is not lost during subsequent chemical and dye treatments, such as those found in the Alcian Blue-Verhoeff’s van Gieson procedure and Movat’s Pentachrome, alcian blue is converted to an insoluble pigment. By exposing alcian blue stained sections to a pre-heated 80°C alkaline alcohol solution, alcian blue is converted to the insoluble pigment, monastral fast blue. Further exposure of the sections to various decolorizers and dyes, fails to change the alcian blue intensity.
QUALITY CONTROL AND CONTROL MATERIALS
1. Appropriate control tissues include colon and small intestine. If lung sections contain bronchi with mucous glands, are used as a control, the tech
RATIONALE FOR USE
Alcian blue is a water soluble, amphoteric copper phthalocyanine, which is most often used as a basic dye. In general, alcian blue has an affinity for sulfated and carboxylated groups of acid mucopolysaccharides, but the specific group demonstrated is dependent on the dye solution pH.
RECOMMENDED FIXATIVES
10% neutral buffered formalin and Bouin’s fixative.
AVOID Chromate Fixatives
MODE OF ACTION (1, 2, 3)
Alcian blue binds electrostatically with the acidic groups in the mucopolysaccharide molecule. The components to be demonstrated are intensely stained if the dyer solution is used at the specific pH at which the reactive groups are fully ionized. To achieve full ionization of the reactive groups, some alcian blue methods begin with a rinsing of sections in the appropriate pH solution prior to staining with the dye solution. In general, strongly sulfated mucins react more consistently at low pH levels. Above a pH of 1.0 their reactions are variable. At a pH of 0.2 only strongly sulfated mucosubstances will be demonstrated, while weak sulfated mucins stain well between a pH of 1.0 - 2.5, and often below 1.0. Carboxylated mucins react with alcian blue at pH 2.5.
Alcian blue produces an intense greenish-blue or teal coloration. To ensure this colour is not lost during subsequent chemical and dye treatments, such as those found in the Alcian Blue-Verhoeff’s van Gieson procedure and Movat’s Pentachrome, alcian blue is converted to an insoluble pigment. By exposing alcian blue stained sections to a pre-heated 80°C alkaline alcohol solution, alcian blue is converted to the insoluble pigment, monastral fast blue. Further exposure of the sections to various decolorizers and dyes, fails to change the alcian blue intensity.
QUALITY CONTROL AND CONTROL MATERIALS
1. Appropriate control tissues include colon and small intestine. If lung sections contain bronchi with mucous glands, are used as a control, the tech
References: 1. Lev R. and Spicer S.S.: Specific staining of sulphate groups, with Alcian blue at low pH. J. Histochem. Cytochem., 12:309, 1964. 2. Sheenhan D.C. and Hrapchak B.B.: Theory and Practice of Histotechnology, 2nd edition. C.V. Mosby Co., 1980