The histochemistry of Alcian blue. Note on the presence and removal of boric acid as the major diluent in alcian blue 8GX.
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Staining of sulphatides in metachromatic leukodystrophy with Alcian blue at high salt concentrations.
Alcian blue combines with purified sulphatide in 1.OM magnesium chloride. In tissue sections from patients with metachromatic leukodystrophy, sulphatide is stained by Alcian blue in 0.8 M magnesium chloride, and the staining can be abolished by prior treatment with chloroform and methanol. The simplicity of the technique, its specificity and ease of interpretation recommend Alcian blue staining at high salt concentrations as a routine method in the diagnosis of metachromatic leukodystrophy.
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Monastral fast blue. Cytochemical properties of a reaction product from Alcian blue stained chromatin.
The staining of chicken erythrocyte nuclei with Alcian blue followed by a treatment with ethanol-NaOH, showed to be highly resistant to acid extraction (5 N HCl) due to the alkaline conversion of Alcian blue into the insoluble pigment Monastral fast blue. It is assumed that the intercalative binding of the planar Monastral fast blue molecule to DNA accounts for its competition effect toward the intercalating fluorochromes acridine organe, ethidium bromine and daunomycin.
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Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride.
Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.
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Chemistry, histochemistry and microscopy of the organic matrix of spicules from a gorgonian coral. Relationship to alcian blue staining and calcium binding.
Alcian blue dye normally binds to polyanionic, polymeric substances. Such structures are often associated with calcium binding portions of the organic matrix in calcifying tissues. The organic matrix of spicules prepared from the gorgonian Pseudoplexaura flagellosa (Houttuyn) is alcianophilic. The dye is very tightly bound to the lipoid portion of the insoluble spicule matrix. No acidic substances (sulfated or acidic polysaccharides or phospholipids) were demonstrable in this material, suggesting an unusual but unknown interaction between dye and substrate. On a microscopical basis, inclusion of Alcian blue (or Ruthenium red) is an essential co-requisite to glutaraldehyde fixation. Without the dye the morphological integrity of the spicule is lost on decalcification. The fragmented matrix is still alcianophilic suggesting that the dye may substitute for material solubilized by the decalcifying agents. Examination of post-decalcification supernatants demonstrate that approximately 13% of the matrix is solubilized on demineralization, releasing 93% of the carbohydrate but less than 20% of the protein. Liberated protein takes the form of peptides ranging from 1100-1500 daltons. The composition of these peptides is a function of the demineralizing agent. Acidic demineralizers produce peptides proportionately high in acidic amino acids, that do not bind calcium. Peptides produced by chelator decalcification appear to bind calcium but other evidence strongly suggests that the binding is due to adsorbed chelator rather than by soluble matrix.
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The molecular biology of histochemical staining by cationic phthalocyanin dyes: the design of replacements for Alcian Blue.
Four cationic copper phthalocyanin dyes are compared with Alcian Blue 8G for specificity of interaction with a range of natural and synthetic polyanions. The CEC system is used to estimate dye-substrate affinities. The results support the hypothesis that the tissue staining pattern of Alcian Blue is determined by (a) exclusion of the dye from polynucleotide stacked base pairs by steric hindrance due to bulky substituents on the chromophore and (b) the presence of pendant quaternary charges whose affinity for negative sites on polyanionic substrates is without regard for the precise chemical nature of the anion. These conclusions, and practical considerations lead to the choice of quaternized Astra Blue 6GLL as a replacement for Alcian Blue (now no longer manufactured). It is superior to Alcian Blue in pH range, and stability in solution. It is easily prepared from a readily available precursor.
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Temperature-dependent staining reaction of chromatin by Alcian blue.
After treatment with Alcian blue at high temperatures, nuclei from chicken blood smears showed an intense staining reaction, which proved to be dependent on the DNA content of chromatin. The possibility that interactions between the phthalocyanine chromophore and unpaired bases accounts for this temperature-dependent staining of chromatin is briefly discussed.
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The usefulness of routine alcian blue staining of corneal specimens.
To determine the diagnostic usefulness of routine staining of corneal buttons with alcian blue, we evaluated 150 consecutive corneal specimens with hematoxylin and eosin and alcian blue stains. Overall, 67% of specimens demonstrated deposition of acid mucopolysaccharide. More specifically, positive alcian blue staining was observed in 88% of specimens with pannus, 83% of specimens with stromal downgrowth, 71% of specimens with stromal scarring, and 67% of specimens with interstitial keratitis. In 80% of these cases, the positive alcian blue stain correlated to pathology noted on hematoxylin and eosin sections. In 20% of cases, we could not explain the reason for the presence of alcian blue staining. Except for macular dystrophy of the cornea, the presence, strength, and location of staining did not provide useful information in terms of making a specific clinical diagnosis. Consequently, alcian blue should be used only in those settings in which the pathologist needs to document or confirm the abnormal presence or absence of acid mucopolysaccharide deposition.
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Orcein-alcian blue staining: a new technique for demonstrating acid mucins in gastrointestinal epithelium.
Orcein-alcian blue staining, a new method for the simultaneous demonstration of sulphated and sialomucins in gastrointestinal epithelium was compared with the standard high iron diamine-alcian blue technique. Sections were oxidised with potassium permanganate and decolourised in oxalic acid. They were stained with orcein for four hours, differentiated for a few seconds in acid alcohol, and then counterstained with alcian blue for half to one minute. There was a good correlation of results between the two methods. Orcein-alcian blue is a safer, cheaper, and quicker method than high iron diamine-alcian blue which can be safely introduced into routine laboratories for the study of acid mucins in the gastrointestinal diseases.
Adenocarcinoma↗
Expression of mucin-2 and correlation with PAS-alcian blue stain in Barrett's esophagus.
BACKGROUND/AIMS: Hematoxylin-eosin stain may fail to show intestinal metaplasia in Barrett's esophagus, and PAS-alcian blue may present difficulties of interpretation due to its more heterogeneous staining. We investigated whether Mucin-2 is a good substitute for PAS-alcian blue in the detection of goblet cells. METHODOLOGY: Biopsy specimens from 47 Barrett's esophagus patients were stained with hematoxylineosin and PAS-alcian blue. Mucin-2 expression was evaluated with monoclonal antibody. The intra- and interobserver agreements about the expression of Mucin-2 and PAS-alcian blue stain were determined by kappa statistics. RESULTS: PAS-alcian blue and Mucin-2 were positive in all cases. Mucin expression was positive in goblet cells and few columnar cells. PAS-alcian blue showed heterogeneous staining. The columnar epithelium and the submucosal glands were also stained. Intra- and interobserver agreement in the identification of intestinal metaplasia was 100%. CONCLUSIONS: Mucin-2 agreed fully with PAS-alcian blue. The intra- and interobserver agreement was perfect, justifying its use in the diagnosis of Barrett's esophagus.
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Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes.
Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions. This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG. For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting. Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results. Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with [(35)S]-sulfate and [(3)H]-glucosamine. The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes. Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels. However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex. The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta.
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Zirconyl hematoxylin vs. alcian blue staining in a case of extramammary Paget's disease.
BACKGROUND: Despite its occasional unavailability, alcian blue has long been the stain of choice for the pathognomonic mucin-secreting cells of extramammary Paget's disease. Zirconyl hematoxylin is a recently developed substitute for alcian blue, which is made from common laboratory materials. METHODS: We have stained sections from three different areas of the same case of extramammary Paget's disease with zirconyl hematoxylin, destained in 7% HCl in ethanol, restained with alcian blue at pH 2.5, destained in 2.5% trifluoroacetic acid in dichloromethane, and restained with zirconyl hematoxylin. RESULTS: Each staining procedure stained the same cells. CONCLUSIONS: Zirconyl hematoxylin is a useful alternative to alcian blue in the diagnosis of extramammary Paget's disease.
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The histochemical specificity of high iron diamine-alcian blue.
The specificity of the High Iron Diamine-Alcian Blue pH 2.5 (HID-AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine-Alcian Blue pH 1.0 demonstrated that complete or almost complete staining of O-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID-AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied 'transitional mucosa' in human colonic diseases. Caution should be used in drawing conclusions from the use of HID-AB 2.5 without confirmatory evidence from other more specific procedures.
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Alcian blue. Now you see it, now you don't.
A brief outline of the history and chemistry of the copper pthalocyanin-based Alcian blue dyes highlights the adverse effects of commercial secrecy and entrepreneurial dishonesty on the use of these valuable histochemical and biochemical reagents in the biological sciences. The acquisition of detailed and validated chemical structures required the avoidable expenditure of much time that otherwise would have been spent on new research. The combination of this chemical knowledge with experiments on isolated polyanions, using critical electrolyte concentration (CEC) techniques, established a molecular recognition paradigm of Alcian blue usage in histochemistry. New reagents (Cuprolinic and Cupromeronic blues) were synthesised to investigate and support the hypothesis that Alcian blue did not intercalate with DNA. They were made with the important additional aim of using them in electron microscopy of polynucleotides (in the case of Cuprolinic blue) and non-nucleotide polyanions, such as sulphated proteoglycans in extracellular matrices (in the case of Cupromeronic blue). The cationic groups of Alcian blue are easily removed in very mild conditions, producing insoluble blue pigment in situ (ingrain dying). This reaction underlies its unique capability to stain in repeated cycles, thus greatly amplifying sensitivity of detection of substrates present in very small amounts in tissues and on electrophoretic strips etc.
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Measurement of tissue disulfide groups by Alcian blue 8GX and atomic absorption spectrophotometry.
The primary purpose of this study was to quantify tissue disulfides with Alcian blue 8GX using atomic absorption spectrophotometry (ASS) to measure the copper moiety of the Alcian blue. The secondary purpose was to determine whether aqueous or alcoholic formalin was a better fixative for this procedure. Sections of mouse renal cortex fixed in 10% neutral formalin (NBF) or in ethanol/formalin/acetic acid (AFA) were stained in 0.5% Alcian blue 8GX in 0.8 M MgCl2 with prior thiosulfation (+Th) or without prior thiosulfation (-Th). The relative contents in the tissues of the copper of Alcian blue, measured by AAS, may be summarized as follows: -Th NBF, 1.15 micrograms Cu/mg tissue; -Th AFA, 1.35 micrograms Cu/mg tissue; +Th NBF, 1.99 micrograms Cu/mg tissue; +Th AFA, 3.25 micrograms Cu/mg tissue. These data indicate that 1) disulfide may be measured by this procedure, and 2) AFA is the preferred fixative for measurement of both disulfides and native sulfates.
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Standardization of staining in glycosaminoglycan histochemistry: alcian blue, its analogues, and diamine methods.
Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results in particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables histochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.
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Basophil counting with a new staining method using alcian blue.
Difficulties in obtaining reproducible and accurate enumeration of circulating basophils with existing techniques have hampered investigation of this infrequent cell population. A new basophil staining method is described that employs alcian blue dye for staining of heparin within basophils at low pH and in the presence of lanthanum ions. Basophil recognition is facilitated by reducing nonspecific nuclear staining. This objective is achieved because of the differences in stability of alcian blue-heparin, alcian blue-nucleic acid, lanthanum-heparin, and lanthanum-mucliec acid complexes. Reduction of pH after staining also favors solubilization of leukocyte cytoplasmic proteins, providing greater contrast between stained and unstained cells by reducing light scattering of the unstained leukocytes . The alcian blue staining method is suitable both for chamber basophil counting and automated basophil counting using continuous-flow sampling and electro-optical detection. The new staining method was evaluated by comparing it with the chamber counting method using toluidine blue in a triple-blind study in which the results of basophil counting by the alcian blue chamber method, alcian blue automated instrument method, and the toluidine blue chamber method were analyzed for reproducibility and compared with an indirect basophil count obtained from a 1000-cell leukocyte differential and a total leukocyte count. Both alcian blue staining methods gave greater reporducibility that toluidine blue and were more accurate, as evidinced by a significantly higher correlation with the indirect basophil count. The improved reproducibility, accuracy, and convenience of this method over existing methods should facilitate the collection of more meaningful information about circulating basophil levels in health and disease.
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Chromatin staining reactions by oxidized p-phenylenediamine and Alcian blue.
After treatment with oxidized p-phenylenediamine or Alcian blue at high temperature, nuclei from chicken and human blood smears showed an intense staining reaction which proved to be dependent on the DNA content of chromatin. The possibility that oxidized p-phenylenediamine binds to phosphate groups and that Alcian blue interacts with bases of denatured DNA is briefly discussed.
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