It is a tetravalent basic (cationic) dye with a copper (Cu2+, coordination 4 of 6, orbital configuration d9 with Jahn–Teller distortion) phthalocyanine nucleus (CuPc) with three or four pendent isothiouronium side chains imparting its bulkiness and positive charges. In order to qualify as an<unk>  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.<unk>  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. 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.<unk>  Blue and the copper phthalocyanine itself are blue, but brominated or chlorinated copper phthalocyanine and sulfonated copper phthalocyanine are green.<unk>  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. 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<unk>  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:<unk>  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<unk>  blue 8G are often stable for some years. Churukian's lab manual gives a recommended shelf life of 6 months. An<unk>  blue solution with a precipitate should be discarded and replaced, not filtered and used. Some dyes sold as<unk>  blue 8G are unstable in solutions at pH 5.6 and above; they precipitate in less than 24 hours. Batches of<unk>  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 pyridine variant of<unk>  blue<unk>  Blue-tetrakis(methylpyridinium) chloride, CAS , CB6503730, PubChem 24860335) is more stable than the original<unk>  blue dyes and may be just as good as a stain. Explanation of staining selectivity Nucleic acids are generally basophilic because they have a very high density of negative charge due to the sugar phosphate backbone. However, in contrast to other basic i.e. cationic dyes,<unk>  blue usually (given the right pH and salt concentrations, and normal temperature and duration in minutes, not hours) preferably stains acidic glycosaminoglycans but not the chromatin and nissel substance, the mechanism of which had been a mystery for a long time and various theories were proposed. Though the presumed basis of the staining is its positive charge attracted to negative structures (e.g. acidic sugars), bulkiness (width 2.5–3 nm, compared to toluidine blue ~0.7 x1.1 nm) makes its diffusion very slow in less permeable parts of the tissue and thus prevent it from staining highly negative yet compact structures such as chromatin and nissl substance. However prolonged staining (few days at 25 °C) or DNA denaturing conditions may allow<unk>  blue to also stain the nucleus. The isolation of the positive charge from the aromatic electron cloud by the intervening methylene bridges makes the localized positive charged regions "hard" ions in contrast to soft ions where the charge is delocalized over the whole aromatic pi cloud. When these hard cations encounter the hard anions e.g. in form of sulfate they form salts without regard for the precise chemical nature of the anion. The resulting salts are highly stable but can be slowly exchanged with high concentration of salts. Washing with water or alkali treatment after staining causes base catalyzed hydrolysis and removal of the pendant positively charged side chains and the resulting compound is Phthalocyanine Blue, which forms a blue water-insoluble dye precipitate. 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.
Alcian blue stain
Wayson stain
Feulgen stain
Auramine phenol stain
Romanowsky stain
Bielschowsky stain
Luxol fast blue stain
Jones' stain