The history,chemistry and modes of action of carmine and related dyes

Carmine has been used in biological staining to demonstrate selectively nuclei, chromosomes or mucins, depending on the formulation. Throughout its history in science, complaints and frustrations have been expressed about dye quality. Inconsistencies in dye quality or identity have prevented thorough understanding of staining mechanisms and have caused many stain solutions to behave unsatisfactorily. The aim of this review is to (1) detail causes of these problems, which are rooted in history, geography and production, (2) offer ways to minimize problems and (3) provide modern explanations for stain behavior. Carmine is a “semi-synthetic” dye, i.e., a complex of aluminum and the natural dye cochineal (carminic acid). Carmine shows considerable batch-to-batch variability. Geography, politics, history, agricultural practices and iconography all contribute to the variability of cochineal. In addition, widely divergent manufacturing methods are used to produce carmine. Also, confusion in terminology has led to mislabeling. Pressure from the food industry for a more satisfactory colorant for acidic foods led to the introduction of a new dye, aminocarminic acid, which could enter the biological market inadvertantly. Improved methods of analysis should help the certification process by the Biological Stain Commission. Further standardization could be achieved by replacing most of the methods of solubilizing carmine. The majority of these methods use heat, which is likely to damage the dye molecule. Fortunately, carmine is readily dissolved by raising the pH of the aqueous solvent above 12, and a new form of the dye, now available commercially, is soluble in water without the need for heat or pH adjustment. Chemical structures and physical properties of carminic acid, carmine, aminocarminic acid and kermesic acid are reviewed. A new configuration for carmine is proposed, as well as possible changes to carminic acid and carmine molecules as a result of decomposition caused by heating. Each of the major classes of carmine-based stains is described as are possible mechanisms of attachment to specific substrates. Glycogen binds carmine through hydrogen bonding, and it is here that carmine decomposed by heat could have the greatest detrimental impact. Nuclei and chromosomes are stained via coordination bonds, perhaps supplemented by hydrogen bonds. Finally, acidic mucins react ionically with carmine. Specificity in the latter case may be due to unique polymeric carmine molecules that form in the presence of aluminum chloride.     

Amyloid from a histochemical perspective. A review of the structure,properties and types of amyloid,and a proposed staining mechanism for Congo red staining

Amyloid is a diverse group of unrelated peptides or proteins that have positive functionality or are associated with various pathologies. Despite vast differences, all amyloids share several features that together uniquely define the group. 1) All amyloids possess a characteristic cross-ß pattern with X-ray diffraction typical of ß-sheet secondary protein structures. 2) All amyloids are birefringent and dichroic under polarizing microscopy after staining with Congo red, which indicates a crystalline-like (ordered) structure. 3) All amyloids cause a spectral shift in the peak wavelength of Congo red with conventional light microscopy due to perturbation of π electrons of the dye. 4) All amyloids show heightened intensity of fluorescence with Congo red, which suggests an unusual degree of packing of the dye onto the substrate. The ß portion of amyloid molecules, the only logical substrate for specific Congo red staining under histochemical conditions, consists of a stack of ß-sheets laminated by hydrophilic and hydrophobic interactions between adjacent pairs. Only the first and last ß-sheets are accessible to dyes. Each sheet is composed of numerous identical peptides running across the width of the sheet and arranged in parallel with side chains in register over the length of the fibril. Two sets of grooves are bordered by side chains. X grooves run perpendicular to the long axis of the fibril; these grooves are short (the width of the sheet) and number in the hundreds or thousands. Y grooves are parallel with the long axis. Each groove runs the entire length of the fibril, but there are very few of them. While Congo red is capable of ionic bonding with proteins via two sulfonic acid groups, physical constraints on the staining solution preclude ionic interactions. Hydrogen bonding between dye amine groups and peptide carbonyls is the most likely primary bonding mechanism, because all ß-sheets possess backbone carbonyls. Various amino acid residues may form secondary bonds to the dye via any of three van der Waals forces. It is possible that Congo red binds within the Y grooves, but that would not produce the characteristic staining features that are the diagnostic hallmarks of amyloid. Binding in the X grooves would produce a tightly packed series of dye molecules over the entire length of the fibril. This would account for the signature staining of amyloid by Congo red: dichroic birefringence, enhanced intensity of fluorescence and a shift in visible absorption wavelength.     

Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.     

Macromolecular changes caused by formalin fixation and antigen retrieval

Although the mechanics of formalin fixation and antigen retrieval have been studied extensively and reviewed periodically, little attention has been directed toward conformational changes in target molecules. Formaldehyde changes the shape of tissue molecules by appending small hydroxymethyl groups to them. These adducts, in turn, can react with other tissue molecules to form crosslinks, or they can participate in a variety of reactions during tissue processing, including formation of imines, ethoxymethyl adducts, and further crosslinks. Under the influence of alcohol dehydration, fixed DNA may fragment and form a variety of depurination products. The situation becomes even more complex with short fixation times because under these conditions, the dehydrating agent used for tissue processing denatures macromolecules in other ways, most notably through rearrangement of molecular shape to move hydrophobic realms outward and hydrophilic areas inward (hydrophobic inversions). How tissue molecules are modified affects the outcome of immunohistochemical staining and prospects for restoration of antigenicity. Immunoreacitivity may be compromised because epitopes are either sterically hidden, but otherwise unaffected, or they have been altered more directly. Enzyme-based retrieval methods are best suited for the former because they literally snip the molecule apart to reveal the portions of interest. Heat-induced retrieval with buffers can demodify affected epitopes by removing adducts and breaking crosslinks. The choice of temperature and pH is usually critical for optimal retrieval. Effective temperatures are directly related to the strength of bonds-higher temperatures are needed to break stronger bonds. The pH of the retrieval solution determines the charge on the tissue molecule; the goal is to create a charge that causes the demodified molecule to assume a near natural conformation. Rational use of these concepts should lead to better control of immunohistochemical reactions.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid.

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

Why fluorescent probes for endoplasmic reticulum are selective: an experimental and QSAR-modelling study

The basis of the selectivity of fluorochromes routinely used to visualize the endoplasmic reticulum (ER) in live cells remains obscure. To clarify this, interactions of living cells with fluorochromes of varied physicochemical properties were analyzed experimentally and numerically using a quantitative structure activity relationship analysis (QSAR). Routine selective ER probes were found to be amphipathic, lipophilic cations with moderate-sized conjugated systems. The moderately lipophilic character permits probe uptake by passive diffusion without nonspecific accumulation in biomembranes. The moderately amphipathic character favors uptake into the ER, perhaps owing to its high concentration of zwitterionic lipid head-groups. The QSAR model rationalizes the impractical character of some ER probes mentioned in the literature, and could permit design of novel ER probes with different emission colors. The possibility of using the QSAR model as a tool to predict the accumulation of xenobiotics in the ER of living cells is illustrated by the localization of certain antipsychotic drugs in cultured cells.