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Alcian Blue

What is Alcian Blue?

It is a type of phthalocyanine compound that is primarily used in the manufacturing of dyes and pigments.

Alcian Blue Synonyms

This compound is also known by other names, such as

  • C.I. 74240
  • Ingrain Blue 1
  • Alcian blue 8GX

Identification Facts

The identification facts of this chemical are as follows:

  • CAS Reference Number: 33864-99-2
  • PubChem Reference Number: 3084579
  • ChemSpider Reference Number: 2341620

Alcian Blue Picture
Picture 1 – Alcian Blue
Source –

Chemical Formula

The chemical formula for this compound is C56H68Cl4CuN16S4.

Physical and Chemical Properties

Go through the main physical and chemical properties of this compound.

Physical state and appearance

It is a solid chemical compound that is mostly found in powder form.


The powder is dark, bluish-violet in color.

Molecular Weight

This chemical’s molecular weight or molar mass is 1298.86 g/mol.

Melting point

This chemical has a melting point of – 98 °C.

Boiling Point

It has a boiling point of 65 °C.

Flash Point

Its flash point is 11 °C.


Alcian Blue is found to dissolve in cold water. However, it is insoluble in methyl alcohol or methanol. All proportion of this chemical is miscible.


It is a stable compound.


It is non-corrosive in glass.


Polymerization of this chemical does not take place.

Alcian Blue Uses

The chemical is mostly used as a cationic dye for staining glycosaminoglycans and acid mucopolysaccharides. Stains are mostly bluish-green and blue.

It is also used in the clinical histology laboratory to demonstrate acid mucins which may be secreted by different epithelial and connective tissue tumors. It is most commonly applied on samples of tissues obtained from the human gastrointestinal tract.

Alcian Blue and PAS Staining Protocol

This method demonstrates the presence of heavily glycosylated proteins or proteoglycans in liver, cardiac, or skeletal tissues. It combines the properties of the Alcian Blue Staining method with the Periodic Acid Schiff Staining method.

Procedure – Firstly, this method involves staining the acidic mucins with Alcian Blue. It helps chemically block the acidic mucins that are PAS-positive and prevent any chemical reaction during the technique. The PAS-positive neutral mucins can be demonstrated in an opposite manner. In the case of any mixture, the dominant moiety can determine the color produced.

Here is how the method needs to be performed:

  • The acidic mucins should be washed in distilled water.
  • Next, they should be stained in Alcian Blue for 15 minutes.
  • After that they should be washed nicely with running tap water for 2 minutes and then rinsed in distilled water.
  • The stained mucins should be treated with periodic acid for 5 minutes and then again washed well with distilled water.
  • Schiff’s reagent should then be used to stain it for 10 minutes.
  • This should then be washed thoroughly with running tap water for 5 minutes.
  • The nuclei should be stained with hematoxylin for 1 minute.
  • Running tap water should be used to wash them for 2 minutes.
  • Acid alcohol should be used for differentiation.
  • A bluing solution should be added after that.
  • The mixture should be washed with running tap water for 5 minutes.
  • The mixture should then be dehydrated, cleared, and mounted.

Results – The method leads to the production of

  • Acidic mucins (blue)
  • Neutral mucins (magenta)
  • Mixtures of the above (blue/purple)
  • Nuclei (deep blue)

Technical Points – Certain technical points should be considered while performing the procedure.

  • Diastase pre-treatment of acidic mucins may be done to remove glycogen before staining in Alcian Blue.
  • Fixation, though unimportant, produces non-specific general PAS positivity when aldehydes like glutaraldehyde are added.

Reagent Formulae – The Reagent Formulae for this chemical is as follows:

1. 1% Alcian blue in 3% aqueous CH3COOH or acetic acid.

2. 0.5% aqueous periodic acid.

3. Schiff’s reagent.

Alcian Blue Cartilage Staining Protocol

This process helps examine the formation of cartilages. It involves the use of a basic water-based dye as a staining agent for acid mucosubstances and acetic mucins found in fixed embryo cartilages.

  1. The embryos from the uterus have to be dissected and washed once with phosphate buffer solution (PBS). The embryos should then be fixed individually at room temperature with Bouin’s Solution for one or two hours. Removal of viscera from embryos during the early stages is not absolutely essential. However, the presence of dark organs and blood makes it important to do so in the case of older embryos.
  2. The extra-embryonic membranes from each embryo are labeled and frozen, then saved for genotyping.
  3. The embryos then have to be rinsed over 24 hours in 15 ml polypropylene centrifuge tubes containing a solution of 70% ethanol and 0.1% Ammonium hydroxide and washed about six to eight times until they appear white.
  4. Each embryo must be washed twice in one hour in a 5% acetic acid solution.
  5. The embryos should then be stained in 0.05% Alcian blue 8GX solution in 5% acetic acid for two hours. Larger embryos will need more time to be stained.
  6. After that, each embryo should be washed twice in one hour with a 5% acetic acid solution.
  7. The embryos should be cleaned in pure methanol twice and transferred to polyethylene scintillation vials. They should be incubated in a solution of benzyl benzoate and benzyl alcohol combined in the proportion of 2:1 for a period of 3 hours and overnight. Care should be taken while using the solution of benzyl benzoate and benzyl alcohol, as it can dissolve plastics.

The solutions used for Alcian Blue Staining Procedure include:

Phosphate Buffer Solution

• 8 g Sodium chloride

• .2 g Potassium chloride

• 1.44 g Sodium phosphate, dibasic

• .24 g Monopotassium phosphate

• 800 ml DDW

Alcian Blue

• 0.05% Alcian blue 8GX

• 5% acetic acid

• Water

Bouin’s Fixative

• 75 ml saturated picric acid

• 25 ml 40% formaldehyde

• 5 ml glacial acetic acid

The embryos should be covered in Bouin’s fixative and stored at room temperature.

Alcian Blue MSDS

The Material Safety Data Sheet (MSDS) for Alcian Blue is as follows:


The material is hazardous if it comes in contact with the skin or eyes or if it is inhaled or ingested. Acute and chronic health damages may be produced by exposure to this chemical. Toxicological data suggest that it is damaging to the mucous membranes. Prolonged or repeated exposure to Alcian Blue can even cause damage to organs.

First Aid Measures

Know the first-aid measures that must be followed upon exposure to this compound.

Eye exposure – In case of eye exposure, the victim’s contact glasses should be removed. The eyes should be washed with ample amounts of water, preferably cold water, for a minimum of 15 minutes. A doctor should be consulted after that to ensure eye safety.

Skin exposure – The affected skin areas should be washed thoroughly with lots of cold water, and then a moisturizer should be applied. The chemical-soaked clothes and shoes should be removed and washed properly before reuse. In case of serious skin contact, the areas should be washed using a disinfectant soap. An anti-bacterial cream should be applied to the contaminated skin. Medical attention is required to ensure the wellness of the skin.

Inhalation – The victim should be taken to a place of fresh air, and tight clothing, if any, should be removed. Artificial respiration and oxygen should be applied if the victim is still not breathing properly. Medical assistance is required to ensure further wellness.

Ingestion – Unless the physician instructs, vomiting should not be induced in the patient. In these cases, an unconscious victim should never be given mouth-to-mouth resuscitation. Any tight clothing like a waistband, belt, tie, or collar should be loosened. A doctor should be called for a checkup.

Fire and Explosion Data

Alcian Blue is inflammable at high temperatures. Combustion of this chemical can produce carbon oxide and carbon dioxide, nitrogen oxides like NO, NO2, sulfur oxides like SO2, SO3, and halogenated compounds. It can also produce certain metallic oxides.

In small fires caused by this compound, dry chemical powder should be used to extinguish the fire. When large fires break out, foam, fog, or water spray should be used to fight the fire. Water jets should not be used for this purpose.

Accidental Release Measures

In small spills, the spilled solid content should be put in a proper chemical waste disposal container using appropriate tools. After that, the area should be washed thoroughly with water. Local and regional regulations regarding chemical disposal should be followed while disposing of chemical wastes.

When spilled amounts are large, a shovel should be used to dispose of the chemical contents. The area should be washed with water, and the sanitary system should be used to dispose of the chemicals.


The directions for handling this compound are as follows:

  • The chemical should be kept away from heat or any sources of ignition.
  • The empty containers can still catch fire. Hence the residue should be evaporated using a fume hood.
  • All containers and equipment should be grounded properly.
  • The chemical dust should not be breathed or inhaled.
  • Protective clothing suitable for dealing with hazardous chemicals should be worn.
  • Proper respiratory gear should be used when ventilation is insufficient.
  • If a person feels unwell, a doctor should be consulted to avail proper medical help.
  • The skin and eyes should be protected from any contact with the chemical.


The chemical containers should be tightly closed and placed in a cool place with adequate ventilation.

Personal Protection

The place storing the chemical should be properly engineered. Adequate ventilation should be supplied, and care should be taken to keep the airborne levels lower than the recommended exposure limits. Proper ventilation is essential to avoid exposure to any chemical dust, mist, or fumes.

While dealing with spills or fires, special protection gear should be used. This should include splash goggles with a face shield, dust respirator, lab coat, proper gloves, and a respirator with appropriate pressure mode approved and certified by OSHA or NIOSH.

Alcian Blue is a chemical frequently used as a stain to study cancer cells. This chemical can also be combined with Van Gieson’s and H&E’s staining methods.



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