Alcian blue stain
Alcian blue is any member of a family of polyvalent basic dyes, of which the Alcian blue 8G has been historically the most common and the most reliable member. It is used to stain acidic polysaccharides such as glycosaminoglycans in cartilages and other body structures, some types of mucopolysaccharides, sialylated glycocalyx of cells etc. For many of these targets it is one of the most widely used cationic dyes for both light and electron microscopy. Use of alcian blue has historically been a popular staining method in histology especially for light microscopy in paraffin embedded sections and in semithin resin sections. The tissue parts that specifically stain by this dye become blue to bluish-green after staining and are called "Alcianophilic". Alcian blue staining can be combined with H&E staining, PAS staining and van Gieson staining methods. Alcian blue can be used to quantitate acidic glycans both in microspectrophotometric quantitation in solution or for staining glycoproteins in polyacrylamide gels or on western blots. Biochemists had used it to assay acid polysaccharides in urine since the 1960s for diagnosis of diseases like mucopolysaccharidosis but from 1970's, partly due to lack of availability of Alcian and partly due to length and tediousness of the procedure, alternative methods had to be developed such as the dimethyl methylene blue method.
John E. Scott, the first person outside the dye industry to crack the chemical secret of this dye, comments:
In addition to its wide use as a stain, Alcian blue has also been used in other diverse applications e.g. gelling agent for lubricating fluids, modifiers for electrodes, charged coating agents etc.
Image:Barrett's mucosa, Alcian blue stain.jpg|thumb|right|250px|Alcian blue stain highlighting the goblet cells of Barrett's esophagus
History
The Monstral blue found to coat the inside of copper vessels used to process phthalic acid derivatives had led to the discovery of Phthalocyanine in 1907. Attracted by the brilliance, stability and insolubility of this chromophore, attempts were made to reversibly modify it so that it would be carried into fabric in a solution and then easily precipitated into an unleachable but finely well dispersed deposit. From this attempt, Alcian blue was first synthesized by the ICI dyestuffs department under N. H. Haddock and C. Wood in the early 1940s and patented in 1947, originally as a textile dye. In 1950 it was used by Steedman as a selective dye for mucins. While the popularity of Alcian blue expanded exponentially, the difficulty involved in its production due to environmentally hazardous intermediate steps made its availability difficult and ICI stopped producing it by 1973. Many of the alternate sources sold similar looking color products with unreliable staining.Prof J. E. Scott worked to decipher the chemistry of Alcian blue, which was known only to the Industry but kept as a tight trade secret. After spending 3 man-years of effort in 1972 he published the structure of Alcian blue and was able to get ICI to confirm it in 1973, incidentally in the same time that ICI also had just stopped producing it.
After the interim crisis since the 1970s when ICI had to stop, there have now been environmentally safe alternative industrial manufacturing of this dye that is supposed to work as well as 8GX but is called 8G since it is made differently. In attempt to answer what was the importance of discovering an alternative method of manufacturing this compound, a company that remanufactured Alcian blue says:
Etymology and capitalization of "Alcian"
The etymology of the name is not certain, and whether to capitalize it is an editorial style choice. Two major scientific and medical dictionaries use the lowercase styling, but there is also worthy support for the capitalized styling. According to Elsevier's dictionary of chemoetymology, the Alcian in Alcian blue might have been coined by contraction of phthalocyanine.". Oxford online dictionary mentions that it was a trademark and also specifiesThis hypothesis is consistent with the name of Alcian green, which is a tetraphenyl-phthalocyanine with copper.
However Prof. J. E. Scott who had cracked the chemistry of Alcian blue himself and later received confirmation from the manufacturer wrote that Alcian was a trademark that ICI preferred to be spelt starting with a capital "A", and he presumes it came from the old English word "halcyon", which has a "romantic and poetic associations with the kingfisher bird and calm seas". Prof. Scott also states that Alcian green was merely a mixture of Alcian blue and Alcian yellow and not a single compound, which is also supported by thin layer chromatography data from various sources e.g. works by another dye expert Prof. R. W. Horobin—one of the two chief editors of the 10th edition of the 75-year-old Conn's Biological Stains Manual published on behalf of the Biological Stain Commission.
Alcian yellow is an azo dye having neither a phthalocyanine ring nor any of the colors of the Kingfisher, but in common with Alcian blue, has hydrolyzable charged thiouronium side-chains and similar stability of the final stained product. On the other hand, there are other phthalocyanine dyes such as Luxol fast blue and Durazol blue, which have not acquired "Alcian" as a part of their names.
Physical properties
Color
The solid Alcian blue is obtained as greenish-black crystals with metallic sheen. The aqueous solution is bright greenish-blue. Though the compound alcian blue itself is unstable the staining it produces is stable and light fast.Paradoxic lack of Metachromasia
Unlike tricyclic thiazines, which are metachromatic due to switching from monomeric to stacked aggregates, Alcian blue is apparently orthochromatic. In common with Astra blue and other similar dyes, this property that it does not change color either by change in concentration or by combination with substrates, makes it very suitable for microspectrophotometry. The apparent lack of metachromasia is not because it is truly orthochromatic but because "it is already fully metachromatic" in aqueous solution.Absorption maximum affected by aggregation
In aqueous solution large numbers of Alcian blue molecules stack together as micelles of very large size, too large to be even dialysed. Thus even at a fairly high dilution, it has an absorption maximum at ~600–615 nm, which is actually not the absorption maximum of a dye monomer but that of the multimer. Since the absorbed light is of yellow orange spectrum, the transmitted/reflected light is perceived by our eye as the complementary color of slightly greenish blue or cyan. In aqueous solution Alcian blues continue to be metachromatic at molar concentrations one hundredth those at which toluidine blue is mainly orthochromatic. Only a very small shoulder of the absorption curve at 670–680 nm represent the monomeric dye, which is usually the minority and becomes even lesser minority in presence of salts. However, when the solvent is DMSO—a non-protic solvent of moderately high dielectric constant, Alcian blue does not aggregate and a big monomeric absorption peak can be well visualized. A similar spectral shift to the longer monomeric peak is also observed when solvents like ethanol is used as a vehicle or when nonionic detergents like Triton X-100 are used, that make exogenous micelles.Molar extinction coefficient
Alcians blue carries Phthalocyanine one of the most highly colored chromophores yet known with a molar extinction of 120,000 i.e. Alcian blue is detectable at half the molar concentration of popular dyes like toluidine blue, tryarylmethanes etc.Solubility
It is water-soluble. When each of the pair of substituents on the pendant group nitrogens are toluyl, the solubility in water at 20 °C is about 9.5% w/w; and similarly a few other solubilities are: 6.0% in absolute ethanol, 6.0% in Cellosolve and 3.25% ethylene glycol, whereas it is practically insoluble in xylene. In relative/partitioning terms, Alcian Blue 8G has a log octanol-water partition coefficient of −9.7, suggesting it is rather water-soluble. Methanol is an acceptable substitute for ethanol as a potential vehicle for Alcian blue, but isopropanol is not, because, within a few hours all of suspended Alcian blue precipitates if isopropanol is tried as a vehicle.Melting point
The sample compound with Merck index number 218 has a melting point of 148 °C.Chemistry
It is a tetravalent basic dye with a copper phthalocyanine nucleus with three or four pendent isothiouronium side chains imparting its bulkiness and positive charges. In order to qualify as an alcian blue family member there has to be at least 2 side chains and the mixtures often have 3 chains in average to qualify as 8G. Four tetramethylisothiouronium groups per molecule are shown in the picture. ICI had claimed an average of about three side chains per molecule, but analyses by Prof Scotts lab suggested between three and four. Most of them are at the 2 positions, as in the formula and sometimes a cartoon representation uses the methylene bridge criss crossing across the bond between these two positions to indicate that it could bind either of these two positions. A large number of isomers, differing in the positions of the cationic groups, are possible. Alcian blue 7GX carries fewer isothiouronium groups than 8GX. Similarly 5GX and 2GX may have even fewer side groups but it was not rigorously proven.The phthalocyanine aromatic nucleus has a large conjugated system with a CBN of 48. However it is the charges on the isothiouronium side groups that still keeps it water-soluble. These side groups can carry bulkier alkyl or aryl substituents rather than the 8x2 methyl groups as in the image given. These groups split off from the macrocyclic ring during the washing at the end of staining or by rather mild conditions or during spontaneous degradation.
The metals in the Phthallocyanine nucleus and substituted groups directly attached to the aromatic nucleus determine colors of the members of the metal phthallocyanine family e.g. Alcian Blue and the copper phthalocyanine itself are blue, but brominated or chlorinated copper phthalocyanine and sulfonated copper phthalocyanine are green.
Alcian Blue has a relatively high solubility in salt solutions and stains slower than other dyes. By changing pH or ambient salt concentrations characteristic staining patterns can be obtained.