Alcian blue stain

Source: Wikipedia, the free encyclopedia.
For the band, see Alcian Blue (band).
Alcian blue stain
Names
Other names
Alcian blue 8GX, Ingrain blue 1, C.I. 74240, "chloromethylated copper phthalocyanine-thiourea reaction products"
Identifiers
3D model (
JSmol
)
ChemSpider
ECHA InfoCard
 100.046.990 Edit this at Wikidata
UNII
  • InChI=1S/C56H68N16S4.4ClH.Cu/c1-65(2)53(66(3)4)73-29-33-17-21-37-41(25-33)49-58-45(37)57-46-38-22-18-35(31-75-55(69(9)10)70(11)12)27-43(38)51(59-46)64-52-44-28-36(32-76-56(71(13)14)72(15)16)20-24-40(44)48(63-52)62-50-42-26-34(19-23-39(42)47(60-49)61-50)30-74-54(67(5)6)68(7)8;;;;;/h17-28H,29-32H2,1-16H3;4*1H;/q+2;;;;;+2/p-4 checkY
    Key: KDXHLJMVLXJXCW-UHFFFAOYSA-J checkY
  • InChI=1/C56H68N16S4.4ClH.Cu/c1-65(2)53(66(3)4)73-29-33- 17-21-37-41(25-33)49-58-45(37)57-46-38-22-18-35(31-75-55 (69(9)10)70(11)12)27-43(38)51(59-46)64-52-44-28-36(32-76- -56(71(13)14)72(15)16)20-24-40(44)48(63-52)62-50-42-26- 34(19-23-39(42)47(60-49)61-50)30-74-54(67(5)6)68(7)8;;;;;/h17- -28H,29-32H2,1-16H3;4*1H;/q+2;;;;;+2/p-4/fC56H68N16S4.4 Cl.Cu/h;4*1h;/qm;4*-1;m
  • InChI=1/C56H68N16S4.4ClH.Cu/c1-65(2)53(66(3)4)73-29-33-17-21-37-41(25-33)49-58-45(37)57-46-38-22-18-35(31-75-55(69(9)10)70(11)12)27-43(38)51(59-46)64-52-44-28-36(32-76-56(71(13)14)72(15)16)20-24-40(44)48(63-52)62-50-42-26-34(19-23-39(42)47(60-49)61-50)30-74-54(67(5)6)68(7)8;;;;;/h17-28H,29-32H2,1-16H3;4*1H;/q+2;;;;;+2/p-4
    Key: KDXHLJMVLXJXCW-XBHQNQODAQ
  • CN(C)C(=[N+](C)C)SCC1=CC2=C(C=C1)C3=NC4=NC(=NC5=C6C=C(C=CC6=C([N-]5)N=C7C8=C(C=CC(=C8)CSC(=[N+](C)C)N(C)C)C(=N7)N=C2[N-]3)CSC(=[N+](C)C)N(C)C)C9=C4C=CC(=C9)CSC(=[N+](C)C)N(C)C.[Cl-].[Cl-].[Cl-].[Cl-].[Cu+2]
Properties
C56H68Cl4CuN16S4
Molar mass 1298.86 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
checkY verify (what is checkY☒N ?)
Micromass cultures of C3H-10T1/2 cells at varied oxygen tensions stained with Alcian blue.

Alcian blue (

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 e.g. Dimethyl methylene blue (DMB or DMMB) method.[2]

John E. Scott, the first person outside the dye industry to crack the chemical secret of this dye, comments:

"Probably no other dyestuff has been applied to such wide variety of problems in biology and medicine. On the other hand, no other dyestuff had such a chequered history as AB.[3]"

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.

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 (ingrained) into an unleachable but finely well dispersed deposit (hence the name "ingrain dyeing"). From this attempt, Alcian blue (Ingrain blue 1) was first synthesized by the ICI dyestuffs department under N. H. Haddock and C. Wood[4] in the early 1940s and patented in 1947, originally as a textile dye.[5][6] In 1950 it was used by Steedman as a selective dye for mucins.[7]
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.[8]

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.[9] In attempt to answer what was the importance of discovering an alternative method of manufacturing this compound, a company (Anatech Ltd, USA) that remanufactured Alcian blue says:

"Alcian blue is highly selective for the tissue substances (given the proper solution pH), and forms insoluble complexes that withstand harsh subsequent treatment (like PAS) without destaining. That is what makes this dye so important. Do any other dyes have this attribute? Yes, two others to be exact, out of thousands listed in the Colour Index and Conn's Biological Stains." These two are 'Alcian yellow' and basic red 18, which are again both equally unavailable and also lack the brilliant contrast of the blue.

Etymology and capitalization of "Alcian"

Alcyone was converted to this bird and caused the calmness of the seas during the Halcyon Days
.

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,[10][11] but there is also worthy support for the capitalized styling (discussed below). According to Elsevier's dictionary of chemoetymology, the Alcian in Alcian blue might have been coined by contraction (and slight alteration) of phthalocyanine.".[12] Oxford online dictionary mentions that it was a trademark and also specifies[13]

"1940s: Alcian perhaps from (phth)al(o)cyan'(ine) with a phonetic respelling".

This hypothesis is consistent with the name of Alcian green, which is a tetraphenyl-phthalocyanine with copper.[14]

However Prof. J. E. Scott who had cracked the chemistry of Alcian blue himself and later received confirmation from the manufacturer (ICI) wrote that Alcian was a trademark that ICI preferred to be spelt starting with a capital "A", and he presumes it came from the

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.[15]

Luxol fast blue and Durazol blue, which have not acquired "Alcian" as a part of their names.[16]

Physical properties

Color

The solid Alcian blue is obtained as greenish-black (or sometimes dark bluish violet[17]) crystals with metallic sheen. The aqueous solution is bright greenish-blue. Though the compound alcian blue itself is unstable (see stability below) the staining it produces is stable and light fast .

Paradoxic lack of Metachromasia

Unlike tricyclic thiazines (e.g.

orthochromatic but because "it is already fully metachromatic" in aqueous solution.[3]

Absorption maximum affected by aggregation

In aqueous solution large numbers of Alcian blue molecules

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 (<108M) 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 (or ethanol water mixture) 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

Gram stain
) 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

isopropanol is not, because, within a few hours all of suspended Alcian blue precipitates if isopropanol is tried as a vehicle.[19]

Melting point

The sample compound with Merck index number 218 has a melting point of 148 °C.

Chemistry

It is a tetravalent basic (cationic) dye with a copper (Cu2+, coordination 4 of 6, orbital configuration d9 with

CuPc) 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(3) 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 (Conjugated bond number) of 48.[18] 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 (e.g. pH above 5.6) 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.

pH controlled staining

At pH 1.0 it stains only sulfated polysaccharides and at pH 2.5 also stains carboxyl group containing sugars such as sialic acids and uronic acids intensify the stain of hyaluronic acids, which would also stain albeit relatively weakly by their half sulfate esters at pH 1.0.[20]

Electrolyte controlled staining

A staining method where at a fixed pH of about 5.5, different critical salt concentration (classically MgCl2, but NaCl, KCl, LiBr are potential alternatives) can be used where the smaller (faster diffusing) salt cation competes with alcian blue to bind to the anionic sites. Target material specific critical electrolyte concentration (CEC) is supposed to selectively identify sulphated, carboxylated and phosphated structures for example as the targets.

Stability

According to John A. Kiernan—one of the editors of the 10th edition of Conn's Biological Stains" 10th ed 2002 published on behalf of the Biological Stain Commission:[21] Alcian blue 8G differs from most other dyes in that it can deteriorate even in the solid state, changing to an insoluble pigment. Acidic solutions of Alcian blue 8G are often stable for some years. Churukian's lab manual [1] gives a recommended shelf life of 6 months. An Alcian blue solution with a precipitate should be discarded and replaced, not filtered and used. Some dyes sold as Alcian blue 8G are unstable in solutions at pH 5.6 and above; they precipitate in less than 24 hours. Batches of Alcian blue that do not form stable solutions cannot be used in Scott's "critical electrolyte concentration" methods for histochemical characterization of different glycosaminoglycans, which require solutions at pH 5.7-5.8 with variable concentrations of MgCl2.

The

CAS 123439-83-8, CB6503730, PubChem 24860335) is more stable than the original alcian blue dyes and may be just as good as a stain.[22]

Explanation of staining selectivity

Phthalocyanine Blue, which forms a blue water-insoluble dye precipitate.[25] The precipitates are so robust that they withstand harsh conditions like PAS or other counterstaining and also dehydration and embedding treatments (in contrast toluidine blue
is partially extracted away during dehydration). This unleachability is the chemical basis of the ingrain dyeing for which AB (Ingrain blue 1) was originally designed by the dye industry.

Manufacturing and purity

The historic Alcian Blue varied so much batch to batch that only the 8GX (e.g. not even the 8GS) batch produced by ICI was later decided to be the biologically useful ones. Commercially available batches usually contained about 49% of the actual dye and rest used to be Sulfate, boric acid, dextrin and other impurities and by various extraction methods up to 80% pure extracts can be made. Actually the dye does not necessary contain all 4 substituents but might contain 2 or 3 of them and have various geometric isomers. But anyhow the manufacture of 8GX by ICI had stopped by Mid-1970s because of environmental hazards and very small lots were available that were received from alternate sources. Only recently Alcian Blue has been re manufactured in bulk using safer procedures but the newer product does not have the suffix X (or S) since the manufacture process (and the exact product composition) is somewhat different.[9]

Material safety

Alcian blue is an eye and respiratory tract irritant. Solid Alcian blue is a combustible powder and should never be handled close to heat or a naked flame. Heating Alcian blue produces toxic fumes of nitrogen compounds. It can react violently if mixed with oxidising materials. The solution of Alcian blue is a skin sensitiser and corrosive (partly due to the acidic pH needed to maintain it unhydrolyzed in solution) and harmful by skin absorption. Most vendor MSDS (Material safety datasheet) mention that effect of ingestion not known or target organ not known. However some do mention that potential target organs are teeth and kidneys.[26]

Uses in Dye Industry

This stain was originally discovered by ICI in the 1940s as a member of the competitive dye industry for the purpose of industrial dying. It was used for some time for staining textiles, leather products and inks. ICI sold thousands of tons of alcian blue and filed multiple patents regarding its manufacturing process to keep its chemistry a tight secret. However ICI had had trouble with the dye's solubility under textile dyeing conditions, and various process changes in manufacturing were made during the 1950s and 1960s.

Uses in biological staining

Drug interference in staining

Uses other than as a dye or stain

In addition to its use as a dye or stain Alcian blue also finds other material science uses.

Adhesive

Alcian blue has been used as an adhesive to help stick glycol methacrylate sections to glass slides (which have negatively charged silicate groups).[27]

Coating agent

Alcian blue carrying a large aromatic surface that can participate in Van der Waal's interaction as well as multiple localized charges. Thus it can be coated onto surfaces and significantly modify surface property and charge. Some cells in culture grow better on surfaces coated with positive charge like

poly-L-lysine or polyornithine or Alcian blue. Alcian blue coated surfaces hold onto the negatively charge glycocalyx so tight that it can even be used to cover a layer of cells and then float it up to peel off the roof ("unroofing") to study the cytoplasmic side of the plasma membrane.[28]

Gelling or lubricating agent

Alcian Blue has been used as a gelling agent for lubricating fluids likely due to the stacking properties of this macrocylic

aromatic
compound.

Electrode component

Another application is "Alcian Blue Modified Carbon Paste Electrode" (ABMCPE), which acts as a highly sensitive redox sensor compared to Bare Carbon Paste Electrode (BCPE) and exhibits strong "promoting" effect and stability.[citation needed]

See also

References

  1. ISBN 9780306442940
    – via Google Books.
  2. ^ Clinical Chemistry March 1989 vol. 35 no. 3 374–379
  3. ^
    PMID 8653492
    .
  4. ^ "Alcian Blue". The I.C.I. Journal: 59–60. March 1948.
  5. ^ GB 586340 
  6. ^ GB 587636 
  7. PMID 24540170
    .
  8. S2CID 35634762
    .
  9. ^ a b c "Three Dyes, Three Dilemmas" (PDF). The Innovator. Anatech Ltd. Autumn 2001. Retrieved 25 February 2018.
  10. ^ Parker, SP, ed. (1994), McGraw-Hill Dictionary of Scientific and Technical Terms (5th ed.). Headword "alcian blue".{{citation}}: CS1 maint: postscript (link)
  11. ^ Elsevier, Dorland's Illustrated Medical Dictionary [online], Elsevier, archived from the original on 2014-01-11, retrieved 2014-05-14.
  12. ^ Alexander Senning. 2007. Elsevier's dictionary of chemoetymology. Amsterdam: Elsevier. p. 11.
  13. ^ "Alcian blue". Oxford English Dictionary. Oxford University Press. Archived from the original on February 26, 2018.
  14. ^ "Alcian green, 37370-50-6". www.chemnet.com.
  15. S2CID 8999433
    .
  16. ^ "Stainsfile - Compare phthalocyanines".
  17. ISBN 9780470586235
    – via Google Books.
  18. ^ a b Horobin, Richard W. (2010). ""Special Stains" - Influence of Dye Chemistry on Staining" (PDF). In Kumar, George L.; Kiernan, John A. (eds.). Special Stains and H & E (2 ed.). Carpinteria, California: Dako. pp. 159–166. Retrieved 2018-02-26.
  19. PMID 6166095
    .
  20. PMID 4137192
    .
  21. ^ Kiernan, John A. "Re: [Histonet] Dry Stains Shelf Life". www.histosearch.com.
  22. S2CID 39534882
    .
  23. ^ Jebaramya, J; Ilanchelian, M; Prabahar, S (December 2009). "Spectral studies of toluidine blue O in the presence of sodium dodecyl sulfate" (PDF). Digest Journal of Nanomaterials and Biostructures. 4: 789–797.
  24. ^ Horobin, Richard W. (2010). "How Do Dyes Impart Color to Different Components of the Tissues?" (PDF). In Kumar, George L.; Kiernan, John A. (eds.). Special Stains and H & E (2 ed.). Carpinteria, California: Dako. pp. 159–166. Retrieved 2018-02-26.
  25. ISSN 1478-4408
    .
  26. ^ "Lifeline Cell Technology MSDS: 1.0% Alcian Blue Stain" (PDF). Lifeline Cell Technologies. 15 January 2008. Retrieved 25 February 2018.
  27. PMID 3520961
    .
  28. ISBN 9780080888163
    – via Google Books.

External links
Retrieved from "https://en.wikipedia.org/w/index.php?title=Alcian_blue_stain&oldid=1218744442"