Alcian Blue

Alcian blue (a histological stain for glucosaminoglycans) is a cationic phthalocyanin which can activate receptor-operated calcium flux in Paramecium and Tetrahymena (Tamm, 1994;

From: The Chlamydomonas Sourcebook , 2009

Extraction Techniques and Applications: Biological/Medical and Environmental/Forensics

M. Lai , B. Lü , in Comprehensive Sampling and Sample Preparation, 2012

3.04.6.3.5 Alcian Blue

Alcian blue is the name given to a family of polyvalent basic dyes. These dyes can react with compounds containing anionic groups such as acid mucosubstances and acid mucins. They color blue because of the presence of copper in the molecule. Several methods that use Alcian blue at different pH values are available. A general method uses an Alcian blue solution at pH 2.5. This Alcian blue solution with numerically low pH value can stain many acidic mucins and acid mucosubstances. The Alcian blue solutions with even lower pH value color only acidic sulfated mucosubstances (pH 1.0) or strongly acidic sulfated mucosubstances (pH 0.4). Alcian blue can be used in combination with PAS, which can therefore simultaneously demonstrate acidic mucins and mucosubstances as well as neutral mucins. To increase staining specificity for various acidic mucins or mucosubstances, preceding digestion methods such as testicular hyaluronidase are also applied. However, these methods are not routinely used at present.

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Alcian blue, pH 2.5

Alcian blue solution, pH 2.5: Dissolve 1   g of Alcian blue (8GX) in 100   ml of 3% acetic acid solution.

Procedures:

1.

Deparaffinize and hydrate to distilled water.

2.

Place in 3% acetic acid solution for 5   min followed by staining in Alcian blue solution, pH 2.5 for 30   min.

3.

Rinse in distilled water and counterstain with filtered nuclear fast red solution for 5   min (optional).

4.

Wash in running tap water for 1   min.

5.

Dehydrate, clear, and mount with resin.

Results: Weakly acidic sulfated mucosubstances, hyaluronic acid, and sialomucins are stained dark blue. Nuclei and cytoplasm are red to pink or pale pink, respectively, if nuclear fast red counterstaining is used.

•

Alcian blue, pH 1.0

Alcian blue solution, pH 1.0: Dissolve 1   g of Alcian blue (8GX) in 100   ml of 0.1   M hydrochloric acid solution.

Procedures:

1.

Deparaffinize and hydrate to distilled water.

2.

Stain in Alcian blue solution, pH 1.0 for 30   min.

3.

Blot sections dry with filter paper without rinsing in water.

4.

Dehydrate, clear, and mount with resin.

Results: Sulfated mucosubstances are dark blue.

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Alcian blue, pH 0.4

Phosphate/hydrochloric acid solution: Add 42   ml of concentrated hydrochloric acid and 13.8   g of monobasic sodium phosphate to 958   ml of distilled water.

Alcian blue solution, pH 0.4: Dissolve 1   g of Alcian blue (8GX) in 100   ml of phosphate/hydrochloric acid solution.

Procedures:

1.

Deparaffinize and hydrate to distilled water.

2.

Place in phosphate/hydrochloric acid solution for 5   min followed by staining in Alcian blue solution, pH 0.4 for 30   min.

3.

Rinse in distilled water and counterstain with filtered nuclear fast red solution for 5   min (optional).

4.

Wash in running tap water for 1   min.

5.

Dehydrate, clear, and mount with resin.

Results: Strongly acidic sulfated mucosubstances are blue. Nuclei and cytoplasm have a red to pink or pale pink color, respectively, if nuclear fast red counterstaining is used.

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Alcian blue, pH 2.5 or pH 1.0–PAS (AB-PAS)

Preparation of solutions: see relevant procedures above.

Procedures:

1.

Deparaffinize and hydrate to distilled water.

2.

Stain in Alcian blue solution, pH 2.5 or pH 1.0 for 30   min. Blot sections with pH 1.0 solution dry.

3.

Oxidize in 1% periodic acid solution for 10   min and wash in distilled water for 5   min.

4.

Place in Lillie's cold Schiff solution for 10–15   min and wash in running tap water for 10–20   min.

5.

Dehydrate, clear, and mount with resin.

Results: Hyaluronic acid and sialomucins, or sulfated mucosubstances are dark blue in Alcian blue pH 2.5 or pH 1.0, respectively. Polysaccharides and neutral mucosubstances are magenta to red.

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Chondrocyte Culture: A Target System to Evaluate

MONIQUE ADOLPHE , ... SYLVIE DEMIGNOT , in In Vitro Methods in Pharmaceutical Research, 1997

1 Alcian blue staining

Alcian blue staining at acidic pH is widely used as an indicator of the presence of sulfated GAG S. 75, 76 This method of proteoglycan evaluation has been especially used for the evaluation of teratogenic effects on micromass cultures of limb-bud mesenchymal cells 66 and has been miniaturized. 77

The cultures are rinsed twice with PBS and fixed in 2% glacial acetic acid in ethanol, then rehydrated through 95% and 70% ethanol. Fixed cultures are stained overnight with 0.5% Alcian blue in 0.1 N HCI and rinsed twice with 0.1 N HCI to remove any unbound dye. A final rinse with distilled water is aspirated to near dryness. Alcian blue is then extracted by 4 M guanidine HCI overnight at 4°C. Absorbance values are read at 600 nm after temperature equilibration. Wells are rinsed twice with distilled water and the same volume of 4M guanidine HCI is added to obtain the blank values, which are subtracted from the gross values.

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Mechanobiology of Embryonic and Adult Tendons

Laura A. Hockaday , ... Catherine K. Kuo , in Tendon Regeneration, 2015

List of Abbreviations

AB

Alcian blue

BAPN

β-Aminopropionitrile

cell-gels

Cell-seeded gels

Col

Collagen type

cm

Centimeter

dyn/cm2

Dyne per square centimeter

ECM

Extracellular matrix

FGF

Fibroblast growth factor

FV-AFM

Force volume-atomic force microscopy

GAG

Glycosaminoglycans

GPC

Golgi-to-plasma membrane carriers

HH

Hamburger Hamilton Stage

Hz

Hertz

JNK

c-Jun N-terminal kinase

kPa

Kilopascal

LOX

Lysyl oxidase

MMP

Matrix metalloproteinases

MPa

Megapascal

μm

Micrometer

μM

Micromolar

mm

Millimeter

mM

Milimolar

scx-GFP

Scleraxis-green fluorescent protein

SHG

Second-harmonic generation

siRNA

Silencing ribonucleic acid

TGF

Transforming growth factor

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Gastrointestinal System

Jean E. Blair , Robert E. Petras , in Differential Diagnosis in Surgical Pathology (Second Edition), 2010

Special Stains and Immunohistochemistry

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Combined Alcian blue and PAS: serves four purposes

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Defines the PAS-negative, glycogen-free basal layer of the esophagus

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Detects dark-blue, globoid goblet cells when intestinal metaplasia is present

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Highlights fungal structures

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Is a useful screening stain for signet cell adenocarcinoma

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Cytokeratin 7 and 20 profiles can help differentiate gastric cardia intestinal metaplasia from esophageal (Barrett) intestinal metaplasia but are rarely indicated clinically

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The "Barrett pattern" shows diffuse (superficial and deep) cytokeratin 7 positivity and bandlike superficial cytokeratin 20 positivity; other patterns usually correlate with gastric-type intestinal metaplasia

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Functional Glycomics

Hiroto Kawashima , in Methods in Enzymology, 2010

5 Histology and Immunostaining

Immunohistochemical staining with Alcian blue is a convenient method to determine the extent of sulfation of mucins in tissues. For this purpose, the Alcian blue staining should be performed at pH 1.0, because it selectively binds to sulfated carbohydrates under this condition ( Spicer et al., 1981). In the following example, serial sections of the colon from WT and GlcNAc6ST-2-deficient (KO) mice are stained with Alcian blue and an antibody against Muc2, a major intestinal mucin, to assess the sulfation of colonic-mucins.

1.

For Alcian blue staining, frozen sections (7   μm thick) of the colon from WT and KO mice are fixed with 10% formaldehyde in PBS for 30   min.

2.

Incubate the fixed sections with 1% Alcian blue 8GX (Sigma) in 0.1 N HCl (pH 1.0) for 2   h, and counterstain the sections with Nuclear Fast Red solution (Sigma).

3.

For immunohistochemical analyses, fix freshly prepared frozen sections (7   μm thick) with 0.5% glutaraldehyde in PBS for 10   min, and block the nonspecific binding sites by incubating with 3% BSA in PBS for 30   min.

4.

Incubate the sections with 4   μg/ml rabbit anti-Mucin 2 pAb (sc-15334, Santa Cruz Biotechnology, Inc.) for 2   h.

5.

After washing with PBS containing 0.1% BSA, incubate the sections with 0.03% hydrogen peroxide in PBS containing 0.1% BSA to inactivate the endogenous peroxidase.

6.

After washing, incubate the sections with horseradish peroxidase-conjugated goat anti-rabbit IgG (H   +   L) (Zymed, diluted 1:400) for 2   h.

7.

After washing, develop the colored reaction product using a metal-enhanced DAB substrate kit (Pierce Biotehnology, Inc.), and counterstain the sections with Nuclear Fast Red solution.

In WT mice, both Alcian blue and the anti-Muc2 antibody clearly stained colonic epithelial cells, indicating that the colonic-mucins are highly sulfated. In the KO mice, the Alcian blue staining of the colon was significantly diminished, while the staining intensity with the anti-Muc2 antibody did not differ from that observed in the WT mice (Fig. 14.3). No obvious further reduction of the staining intensity with Alcian blue was found in the GlcNAc6ST-1 and GlcNAc6ST-2 double-null mice (data not shown). These results indicate that the sulfation of colonic-mucins is largely mediated by GlcNAc6ST-2.

Figure 14.3. Expression of sulfated carbohydrates and Muc2 in the colon of WT and KO mice. Frozen sections from WT and KO mice were stained with Alcian blue (pH 1.0), or anti-Muc2 pAb. Bar, 50   μm.

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Constitutive Activity in Receptors and Other Proteins, Part A

Frederick S. Kaplan , ... Eileen M. Shore , in Methods in Enzymology, 2010

4.5 Alcian blue staining

Differentiation into cartilage can be evaluated by Alcian blue staining of proteoglycans in the extracellular matrix. Micromass cultures are fixed with Kahle's fixative (28.9% [v/v] EtOH; 0.37% formaldehyde; 3.9% [v/v] acetic acid) and stained with 1% Alcian blue in 0.1  N HCl overnight. Excess dye is removed by washing with water, and cultures are dried before photographs are taken. For quantification of incorporated Alcian blue into the proteoglycan-rich extracellular matrix, cultures are incubated with 6 M guanidine hydrochloride overnight, followed by photometric measurement at OD595.

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Malignant Neoplasms of the Salivary Glands

Simion I. Chiosea , Lester D.R. Thompson , in Head and Neck Pathology (Third Edition), 2019

Ancillary Studies

Histochemical and Immunohistochemical Findings

The pseudocysts/pseudolumina are positive for PAS and Alcian blue ( Fig. 13.31) and react with antibodies to basement membrane components such as laminin and type IV collagen. The epithelial cells are positive for low-molecular-weight keratins (including CK5/6), EMA, and CD117; the myoepithelial cells are positive for markers such as calponin, smooth muscle actin (SMA), p63, p40, and S100 protein (Figs. 13.33 and 13.34).

Molecular Findings

AdCC shows MYB activation, commonly by MYB-NFIB fusion in 40% to 60% of cases.

Fine Needle Aspiration

AdCC contains intensely metachromatic spherical hyaline globules (resembling pink "gum balls"), which represent the reduplicated basement membrane material intimately surrounded by the neoplastic cells (Fig. 13.35). This is best seen on air-dried preparations (they stain pale blue or pink on Papanicolaou [Pap] stain). The globules do not have a fibrillar or feathery edge, as seen in pleomorphic adenoma (PA). There are often rounded stromal structures between the nests of tumor cells. The tumor cells are cohesive, often showing high cellularity and cellular overlap of cells that have little pleomorphism (Fig. 13.36). The nuclei are often peg-shaped, identified in cells with a high nuclear-to-cytoplasmic ratio. The chromatin is heavy and coarse in distribution. On FNA, it may be difficult to separate an AdCC from a PA.

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Nonsquamous Pathologic Diseases of the Hypopharynx, Larynx, and Trachea

Margaret S. Brandwein-Gensler , ... Douglas R. Gnepp , in Diagnostic Surgical Pathology of the Head and Neck (Second Edition), 2009

Special Studies

The glandular elements often produce mucinophilic material that stains with Alcian blue and are hyaluronidase resistant. The epithelial elements, as well as the spindle cells, may express low and high molecular weight cytokeratin and epithelial membrane antigen. Many monophasic SSs also express cytokeratin. The spindle cells, but not the epithelial cells, express vimentin.

The t(X;18)(p11.2;q11.2) balanced translocation, which brings together the SYT and SSX genes, is characteristic for SS and detectable in more than 90% of tumors. This translocation may be detected by fluorescence in situ hybridization or the chimeric SYT/SSX transcript may be detected by reverse transcriptase polymerase chain reaction. 623

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Epithelial Neoplasms of the Esophagus

JONATHAN N. GLICKMAN , ROBERT D. ODZE , in Surgical Pathology of the GI Tract, Liver, Biliary Tract, and Pancreas (Second Edition), 2009

SPECIAL STUDIES

A mucoid matrix that stains positive for periodic acid–Schiff (PAS) and alcian blue may be present in the cribriform spaces of some tumor cell nests. By immunohistochemistry, basaloid carcinomas typically show weak staining for broad-spectrum keratins (in a membrane-type pattern) and P63, and absence of staining for neuroendocrine markers neuron-specific enolase (NSE) and S100. The basal cells stain strongly for CK14 and CK19. 89–91 Some tumors show focal actin and vimentin positivity in the basaloid cells, and carcinoembryonic antigen positivity in the pseudoglandular spaces. At the molecular level, basaloid carcinomas are similar to conventional squamous cell carcinomas, particularly with regard to the frequency of alterations of the p53 and retinoblastoma (Rb) tumor suppressor genes. 92

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Neuronal and Glioneuronal Neoplasms

Daniel J. Brat MD, PhD , Arie Perry MD , in Practical Surgical Neuropathology: A Diagnostic Approach (Second Edition), 2018

Ancillary Diagnostic Studies

Although there are no highly specific stains associated with this mostly morphology-based diagnosis, a histochemical stain for Alcian blue can sometimes highlight the multinodular intracortical pattern (see Fig. 10.11B). DNTs are complex neoplasms composed of glial and neuronal cells. The stellate and piloid astroglial elements are GFAP immunoreactive (Fig. 10.11D), while mature neurons label variably for class III β-tubulin, NSE, neurofilament, NeuN, and synaptophysin. 143,150,151 Synaptophysin staining of neuropil-like matrix components, especially around small blood vessels, is also common. Oligodendrocyte-like cells of the specific glioneuronal element are generally immunoreactive for OLIG2 and S-100 protein but are GFAP negative. Subsets of these cells stain with antibodies to class III β-tubulin and NeuN, suggesting limited neuronal differentiation. MIB-1 labeling indices do not usually exceed 1% to 2%, and are often well below 1%, but values of up to 8% have been noted by some observers.

Ultrastructural studies have yielded conflicting data regarding the differentiation potential of the oligodendrocyte-like cells common to DNTs. 152 Some show neuronal features (neurite-like processes, clear and dense core vesicles, and axosomatic synaptic complexes), while others show oligodendroglial differentiation (such as rudimentary lamellar membrane formations and perineuritic infoldings). Such cells may, therefore, constitute a population capable of divergent glioneuronal differentiation.

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