At least four distinct blue cationic phthalocyanine dyes sold as "alcian blue" raises the question: what is alcian blue?

We investigated physicochemical characteristics of dye lots sold as "alcian blue" using the Biological Stain Commission (BSC) precipitation test, differential scanning calorimetry, high performance liquid chromatography, thin layer chromatography and UV/visible spectroscopy. Four blue phthalocyanine dyes were detected in 11 commercial dye lots. These four included the original ingrain blue 1 CI 74000 dye and the dye sold with the name "alcian blue pyridine variant"; we discuss also the possible identity of the additional two dyes. A proposed extension to the BSC analytic scheme is presented that could distinguish three categories of commercial alcian blue dyes from each other and from the original alcian blue 8G.

Certification procedures used by the Biological Stain Commission for eriochrome cyanine R (C.I. 43820, Mordant blue 3).

Eriochrome cyanine R (C.I. 43820, Mordant blue 3), also known as chromoxane cyanine R and solochrome cyanine R, has been used as a biological stain since 1957. In conjunction with ferric ions, it provides selective blue coloration of the nuclei of cells in methods procedurally similar to commonly used progressive or regressive hemalum (aluminum-hematoxylin) stains. Eriochrome cyanine R also is used to stain the myelin sheaths of axons in nerve tissue; the results are visually similar to those in sections stained with luxol fast blue MBS (C.I. 74180, solvent blue 38) with selective blue coloration of myelin and erythrocytes. Eriochrome cyanine R is an article of commerce with many uses in industrial coloration and analytical chemistry; it can be used instead of either hematoxylin or luxol fast blue MBS, especially in the event of a shortage of either of the latter compounds. The Biological Stain Commission (BSC) will certify batches of eriochrome cyanine R that meet the criteria set out in this document. The criteria include satisfactory UV/visible spectra at pH 4 and pH 12 - 13, a dye content not less than 40% and not greater than 52% (calculated as the color acid; equivalent to 46 - 59% of the trisodium salt), and satisfactory performance in three staining methods: regressive for nuclei, progressive for nuclei and regressive for myelin.

Revised tests and standards for Biological Stain Commission certification of alcian blue dyes.

Alcian blue dyes are copper phthalocyanines with a variety of cationic side chains; they are useful for staining carbohydrate polyanions while avoiding staining of nucleic acids. The properties of the original alcian blue and of similar dyes with published chemical structures are reviewed here. Variation among samples submitted to the Biological Stain Commission (BSC) for certification has led to the recognition of two types of commercially available alcian blue at this time. The designation "alcian blue 8G or equivalent" is reserved for dyes that resemble alcian blue 8GX manufactured in the 1960s (CI 74240; ingrain blue 1). These dyes react with alkali to form an insoluble pigment that cannot be re-dissolved in acid. The name "alcian blue variant" is for similar dyes that do not form insoluble pigments; an alkali-induced precipitate, if formed, re-dissolves with acidification. For certification by the BSC, both types of alcian blue must dissolve in 3% acetic acid to make a 1% solution (pH close to 2.5), which must provide selective coloration of intestinal mucus, cartilage and mast cells, but not of nuclei. After alcian blue staining and treatment with 0.03 M Na2CO3 or Li2CO3 to convert the bound dye to a pigment, the Feulgen stain for DNA is applied. Dyes to be certified as alcian blue 8G or the equivalent must resist extraction by the 5 M HCl used in the Feulgen reaction. Dyes to be certified as alcian blue variant are not required to be convertible to acid-insoluble pigments, but they must dissolve easily in water at pH 5.7 containing 0.5 M magnesium chloride and the dye must remain in solution for at least 24 h. A critical electrolyte concentration (CEC) staining test also is described; this must be passed for certification of an alcian blue variant. Successful CEC staining is also a desirable property of alcian blue 8G or equivalent, but not essential for certification of an otherwise satisfactory batch. The spectrophotometric criteria for alcian blue dyes also are revised; a wider range of absorption maximum (605-634 nm) is allowed. The dye powders used in published staining techniques with the original alcian blue 8G were 40-60% dye, but some modern alcian blue dyes have dye content as high as 90%. The BSC's assay for dye content is not a criterion for certification, but it should influence the amount of dye to include in a staining solution.

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA, and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al3+ ions for each hematein molecule, at pH 2.0-2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular "background color." Such solutions contain a red cationic dye-metal complex and an excess of Al3+ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5-8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0-3.5. Staining by hemalum at pH 2.0-3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pKa ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al2(SO4)3 followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0-3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al3+ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al2(SO4)3 solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.

A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule.

The simple polychrome methylene blue (PMB) staining procedure for blood or tissue smears from dead animals (M'Fadyean reaction) established in 1903 remained accepted as a highly reliable, rapid diagnostic test for anthrax for six decades while that disease was still common in livestock throughout the world. Improvements in disease control led to anthrax becoming rare in industrialized countries and less frequent in developing countries with the result that quality controlled, commercially produced PMB became hard to obtain by the 1980s. Mixed results with alternative methylene blue-based stains then led to diagnosis failures, confusion among practitioners and mistrust of this procedure as a reliable test for anthrax. We now report that, for laboratories needing a reliable M'Fadyean stain at short notice, the best approach is to have available commercially pure azure B ready to constitute into a solution of 0.03 g azure B in 3 ml of 95% ethanol or methanol to which is then added 10 ml of 0.01% KOH (0.23% final azure B concentration) and which can then be used immediately and through to the end of the tests. Stored in the dark at room temperature, the shelf life is at least 12 months. Smears should be fixed with ethanol or methanol (95-100%), not by heat, and the stain left for 5 min before washing off for optimum effect.

Anatomy of the temporal lobe.

Only primates have temporal lobes, which are largest in man, accommodating 17% of the cerebral cortex and including areas with auditory, olfactory, vestibular, visual and linguistic functions. The hippocampal formation, on the medial side of the lobe, includes the parahippocampal gyrus, subiculum, hippocampus, dentate gyrus, and associated white matter, notably the fimbria, whose fibres continue into the fornix. The hippocampus is an inrolled gyrus that bulges into the temporal horn of the lateral ventricle. Association fibres connect all parts of the cerebral cortex with the parahippocampal gyrus and subiculum, which in turn project to the dentate gyrus. The largest efferent projection of the subiculum and hippocampus is through the fornix to the hypothalamus. The choroid fissure, alongside the fimbria, separates the temporal lobe from the optic tract, hypothalamus and midbrain. The amygdala comprises several nuclei on the medial aspect of the temporal lobe, mostly anterior the hippocampus and indenting the tip of the temporal horn. The amygdala receives input from the olfactory bulb and from association cortex for other modalities of sensation. Its major projections are to the septal area and prefrontal cortex, mediating emotional responses to sensory stimuli. The temporal lobe contains much subcortical white matter, with such named bundles as the anterior commissure, arcuate fasciculus, inferior longitudinal fasciculus and uncinate fasciculus, and Meyer's loop of the geniculocalcarine tract. This article also reviews arterial supply, venous drainage, and anatomical relations of the temporal lobe to adjacent intracranial and tympanic structures.

Certification procedures for sirius red F3B (CI 35780, Direct red 80).

Sirius red F3B (CI 35780, Direct red 80) is a polyazo dye used principally in staining methods for collagen and amyloid. For certification by the Biological Stain Commission, a sample of the dye must exhibit an absorption spectrum of characteristic shape with a maximum at 528-529 nm, a small shoulder near 500 nm and narrow peaks at 372, 281-282 and 230-235 nm. Spot tests (color changes with addition of concentrated H(2)SO(4) or HCl and subsequent dilution or neutralization) also are applied. The dye must perform satisfactorily in the picro-sirius red method for collagen by providing red staining of all types of collagen with yellow and green birefringence of fibers. Llewellyn's alkaline sirius red method applied to tissue known to contain amyloid must show red coloration of the products with green birefringence. Dye content, which does not influence significantly the staining properties of sirius red F3B, is not assayed.

A system for quantitative evaluation of fixatives for light microscopy using paraffin sections of kidney and brain.

A numerical scoring system is presented for evaluating structural fixation of certain mammalian tissues for light microscopy. Small pieces of rat's kidney and brain, tissues for which artifacts of fixation are well documented, were fixed in various fluids. Random code numbers hid their identities, and paraffin sections were stained to show nuclear chromatin, cytoplasm and extracellular materials. Two sections of each specimen were examined and awarded scores according to described schemes, for microanatomical and cytological fixation. The assessment was confined to preservation of structure; chemical changes were not taken into account. When the code was broken, the scores for both sections of each specimen from each fixative were added. The scores obtained (24 best to eight worst) are generally comparable to the grades (I-V) given for traditional fixatives by JR Baker. The criteria for quality of fixation were defined more explicitly than those for Baker's grading method, which used mouse testis as the test object. Assessments are presented for several traditional fixatives and for four zinc-formaldehyde mixtures. The scoring system should be useful for evaluating newly developed fixatives for animal tissues for light microscopic examination of paraffin sections. In an evaluation of four traditional fixatives and four zinc-formaldehyde mixtures, variation among three different observers was only +/-1 point on either side of the median score for microanatomical or cytological preservation by any of the eight fixatives. This approach has certain limitations, notably that the criteria for cytological fixation do not include the preservation of chromosomes or specific cytoplasmic organelles.

News from the Biological Stain Commission.

In the three earlier editions of News from the Biological Stain Commission (BSC), under the heading of "Regulatory affairs," the BSC's International Affairs Committee reported on the work of Technical Committee 212, Clinical Laboratory Testing and in Vitro Diagnostic Test Systems of the International Standards Organization (ISO/TC 212) and its working groups, WG 1, WG 2 and WG 3. In this issue of News from the BSC, H.O. Lyon provides information from the annual meeting of ISO/TC 212 that took place June 2-4, 2008 in Vancouver, British Columbia, Canada. In addition, under the heading of "Certification," J.A. Kiernan examines the certification procedure for thionine used by the BSC laboratory in Rochester, NY.

Indigogenic substrates for detection and localization of enzymes.

Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic histochemistry. Indoxyl substrates also are used to localize enzyme activities in non-indigogenic techniques. In indoxyl-azo methods, the released indoxyl couples with a diazonium salt to form an azo dye. In indoxyl-tetrazolium methods, the oxidizing agent is a tetrazolium salt, which is reduced by the indoxyl to an insoluble coloured formazan. Indoxyl-tetrazolium methods operate only at high pH; the method for alkaline phosphatase is used extensively to detect this enzyme as a label in immunohistochemistry and in Western blots. The insolubility of indigoid dyes in water limits the use of indigogenic substrates in biochemical assays for enzymes, but the intermediate indoxyl and leucoindigo compounds are strongly fluorescent, and this property is exploited in a variety of sensitive assays for hydrolases. The most commonly used substrates for this purpose are glycosides and carboxylic and phosphate esters of N-methylindoxyl. Indigogenic enzyme substrates are among many chromogenic reagents used to facilitate the identification of cultured bacteria. An indoxyl substrate must be transported into the organisms by a permease to detect intracellular enzymes, as in the blue/white test for recognizing E. coli colonies that do or do not express the lac-z gene. Secreted enzymes are detected by substrate-impregnated disks or strips applied to the surfaces of cultures. Such devices often include several reagents, including indigogenic substrates for esterases, glycosidases and DNAse.

Revised procedures for the certification of carmine (C.I. 75470, Natural red 4) as a biological stain.

Carmine is one of the original dyes certified by the Biological Stain Commission (BSC). Until now it has lacked both an assay procedure for dye content and a means to positively identify the dye. The methods for testing carmine in the laboratory of the BSC have been revised to include spectrophotometric examination at pH 12.5-12.6 to determine that the dye is carmine (lambda(max)=530-335 nm). The maximum absorbance of a solution containing 100 mg of dye per liter of water, adjusted to pH 12.5-12.6, which provides a relative measure of dye content, should lie in the range 1.2 to 1.8. If the dye is not carmine, spectrophotometry at pH 1.9-2.1 shows whether it is carminic acid (lambda(max)=490-500 nm) or 4-aminocarminic acid (lambda(max)=525-530 nm). The latter two dyes, which are also called carmine when sold as food colorants, have physical properties different from those of true carmine. The functional tests for carmine as a biological stain are Orth's lithium-carmine method for nuclei, Southgate's mucicarmine method for mucus, and Best's carmine method for glycogen.

The E protein HEB is preferentially expressed in developing muscle.

Myogenesis is regulated by the MyoD class of myogenic regulatory factors (MRFs). These basic helix-loop-helix transcription factors dimerize with E proteins to bind conserved E-box sequences in the promoter regions of muscle-specific genes. Perhaps due to their expression in a wide array of tissues, the specific interactions of E proteins with different MRFs have been largely ignored. Likewise, the expression of E proteins in muscle tissue remains mostly uncharacterized. We investigated the expression of the E proteins HEB, E12, and E47 in rat L6 myoblasts, which express only embryonic and fast (2X) myosin heavy chains (MyHCs) in vitro, C2C12 myosatellite cells, and a number of muscle tissues, to determine whether myosin heavy chain diversity is mirrored by diversity in E protein or MRF expression. Although L6 and C2C12 myotubes demonstrate strong expression of embryonic and 2X (fast) MyHCs, immunofluorescence demonstrated the additional expression of type 1 (slow), 2A, and 2B MyHCs in the C2C12 cell line. Immunofluorescence and western blot analyses show that HEB was expressed in differentiating L6 myoblasts, C2C12 cells, and neonatal rat primary myotubes. In contrast, E12 and E47 expression was not detected in either cell line or in any adult muscle tissue examined. These data strongly implicate HEB in the development of skeletal muscle. However, because HEB is expressed in L6 myoblasts, C2C12 myosatellite cells, and neonatal hindlimb muscles, it is unlikely to be involved in a fiber type-specific manner, and may have a more general role in differentiation of myotubes.

Hexazonium pararosaniline as a fixative for animal tissues.

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a "fixative" action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.